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Executive Summary

Our research activities in Year 3 of CAN-6 involved 19 projects that fell under four themes:
1) Biomolecules of Life and Microbial Processes in Analog Settings, 2) Experimental Studies of Paleoenvironmental and Biological Proxies, 3) Archean and Proterozoic Environments and Biosphere, and 4) Developing new Astrobiology Technologies.

Theme 1: Biomolecules of Life and Microbial Processes in Analog Settings

Six projects were pursued under this theme. In Project 1A: Ground control experiments for the OREOcube mission, Co-I’s Pascale Ehrenfreund and Richard Quinn studied the effects of solar and cosmic radiation on organic molecules and potential biomarkers when in contact with minerals and inorganic surfaces. Ground control experiments were performed in preparation for the OREOcube mission. Low-earth-orbit and Mars simulation experiments of organic-inorganic thin films showed intriguing effects and photo-kinetics, and will serve as baseline data for in-flight measurements on the International Space Station (ISS). In Project 1B: Recovery of mineral-associated amino acids, Co-I Pascale Ehrenfreund focused on a key issue of life detection, where amino acid recovery from mineral matrices is a limiting factor for strategies and instrumentation designed for planetary life-detection missions. Her group performed amino acid spiking experiments of a range of Mars-relevant minerals, followed by water extraction procedures, in order to determine recovery rates and efficiencies. These data provide a starting point for how to improve life-detection strategies based on amino acid quantification, and will help to develop advanced methods of organic extraction from minerals.

Co-I’s Eric Roden and Eric Boyd pursued two projects that investigated microbial iron cycling in systems that are analogs to early Mars. In Project 1C: Analysis of dissimilatory microbial iron-reducing microbial communities in Chocolate Pots hot spring, Yellowstone National Park, geochemical (bulk Fe redox speciation, mineralogy, and stable Fe isotope compositions), microbiological (in vitro incubation and enrichment culturing), and genomic (16S rRNA and shotgun metagenomic) analyses were done to determine the distribution of dissimilatory microbial iron reduction potential in Chocolate Pots hot spring. In Project 1D: Comparative genomic analysis of chemolithotrophic Fe(II)-oxidizing bacteria, analysis of new and existing genomes of chemolithotrophic Fe(II)-oxidizing bacteria was done to assess the capability of organisms to grown via oxidation of insoluble Fe(II)-bearing silicate minerals. The goal is to better understand extracellular electron transport by Fe(II)-oxidizing bacteria, and to provide a list of candidate genes for further experimental and genomic studies.

In Project 1E: Studies of early-evolved enzymes in modern organisms may reveal the history of Earth’s ambient temperature over geological time, Co-I Bill Schopf focused on nucleoside diphosphate kinase (NDK), a universally present enzyme that catalyzes the transfer of a phosphate from a nucleoside triphosphate (e.g., ATP) to a nucleoside diphosphate (e.g., ADP). Comparative analyses were made of the thermal stability of reconstructed ancient forms of the enzyme in various early- and late-evolving lineages of phototrophs (e.g., photosynthetic bacteria, cyanobacteria, algae, higher plants), which may provide insight into the temperature-history of Earth’s photic zone environment over geological time.

The formation process of Fe-Mn-nodules may help us to understand the Fe-oxide nodules and elements associated with the Fe-oxide nanophases on Mars, and this was the focus of Project 1F: Study of modern Fe-Mn nodules in Green Bay sediments as analog to the Fe-nodules and Fe-oxyhydroxide minerals on Mars, led by Co-I Huifang Xu. The origin of nano-phase minerals, as well as incorporation mechanism of As, P, Si, Ba, Co, Ni, and Zn, were investigated in freshwater nodules from Green Bay (WI) by in-situ XRD, Scanning Electron Microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Z-contrast imaging, and ab initio calculations using density functional theory (DFT). Combining Z-contrast images and TEM-EDS revealed that the arsenate AsO₄ tetrahedron may be preferentially retained at the proto-goethite surface through tridentate attachment. Similar mechanisms likely explain the high association of P, Si and S with the FeOOH nano-phases, and this understanding will provide an interpretive framework for studying trace element distributions in ancient Fe-Mn deposits.

Theme 2: Experimental Studies of Paleoenvironmental and Biological Proxies

Four projects were pursued under this theme. In Project 2A: Application of the ¹³C-¹⁸O clumped isotope thermometer to the ancient Earth, led by PI Clark Johnson, the clumped isotope thermometer was applied to the late Archean Campbellrand carbonate platform in South Africa, which experienced only very low grades of burial temperatures and metamorphism, up to ~170 oC. Comparison of clumped isotope temperatures for calcite and dolomite confirm experimental studies that indicate relatively fast re-setting of clumped isotope temperatures for calcite relative to dolomite. Despite slower re-setting kinetics for dolomite, however, dolomite temperatures of ~90 oC indicate that even this mineral does not retain primary precipitation (ocean) temperatures for terranes buried for 1-2 b.y. In contrast, the thermal history of carbonates on Mars are likely to have been much lower, and these results indicate that clumped isotope thermometry is likely to be successful on Mars samples.

Continuing the focus on carbonates as a proxy for ancient environmental conditions, Co-I Chris Romanek, in Project 2B: Cation content of carbonate minerals as a pCO₂ proxy: Inorganic synthesis experiments, studied the effect of pCO₂ and temperature on the reaction mechanisms and kinetics for carbonate minerals. A suite of laboratory experiments was initiated to characterize the effect of pCO₂ on cation content (i.e., Ca, Mg and Fe) and Mg-isotope compositions of carbonate minerals. A novel chemo-stat system was constructed and tested to evaluate the control of pCO₂ (<1 to >90% CO2) on the Mg-content and isotope composition of calcite precipitated from solutions held at a steady state chemical composition. A novel new approach included obtaining FTIR spectra on the solids to better understand the role hydration water plays in the incorporation and isotope fractionation of Mg in the calcite lattice.

In Project 2C: Investigation of the role of polysaccharide in the dolomite growth at low temperature by using atomistic simulation, Co-I Huifang Xu explored the conditions required to produce dolomite, a common carbonate in the geologic record of the early Earth and possibly Mars. Dehydration of water from surface Mg²⁺ is most likely the rate-limiting step in dolomite growth at low temperature. The role of polysaccharide in this step was studied using classical molecular dynamics (MD) calculations. MD simulations revealed that oligosaccharide (a model for polysaccharides) can decrease the dehydration barrier of surface Mg²⁺ by about 1 kcal/mol. The hydrophobic space near the surface created by the non-polar –CH groups of the oligosaccharide in the bridge conformation may be the reason for the observed reduction in the dehydration barrier. These results support the idea that simply the presence of dolomite in a rock sequence may be a biosignature.

Co-I Max Coleman continued work on carbonate-associated-sulfate in nodular concretions under Project 2D: Carbonate-associated sulfate (CAS) as a tracer of ancient microbial ecosystems. His group extracted and measured the sulfur isotope composition of total reduced sulfur in the system, which should approximate the hydrogen sulfide that is oxidized to sulfate by dissolved atmospheric oxygen. The isotopic data can be described by a mixing trend of sulfur and oxygen isotope data with sulfate-reduction and sulfide oxidation end-members. Ultimately, the Δ¹⁷O values, reflecting variations in mass-dependent O isotope fractionation, of extracted sulfate may prove to be a proxy for paleo-atmospheric oxygen levels. Currently these results are being confirmed on the very high-resolution mass-spectrometer at UCLA.

Theme 3: Archean and Proterozoic Environments and Biosphere

Seven projects were pursued under this theme. In Project 3A: Apatitic latest Precambrian and Early Cambrian fossils provide direct evidence of concentrations of environmental oxygen, Co-I Bill Schopf studied patterns of substitution of rare earth elements (such as samarium) in fossil-permineralizing Ediacaran through Early Cambrian apatite, an approach that is designed to provide a proxy for the presence or absence of dissolved oxygen in phosphate-precipitating waters. Laboratory studies suggest that this may provide the first paleobarometer that may quantify shallow-water O₂-concentrations immediately before and during the Cambrian explosion of life.

PI Clark Johnson and Co-I Brian Beard led two studies of Archean iron-rich sediments (jaspers and Banded Iron Formations, or “BIFs”) that identified a major role for microbial iron reduction, and constrained marine oxygen contents. In Project 3B: Biologically recycled continental iron is a major component in banded iron formations, the very large 2.5 Ga BIFs (Brockman Iron Formation, Australia) were studied for both stable Fe isotopes and radiogenic Nd isotopes, a novel combination that uniquely distinguishes between hydrothermal and microbial sources of iron. High-ε_Nd and -δ⁵⁶Fe signatures in some BIF samples record a hydrothermal component, but correlated decreases in ε_Nd and δ⁵⁶Fe values reflect contributions from a continental component. The continental Fe source is best explained by Fe mobilization on the continental margin by microbial dissimilatory iron reduction (DIR) and confirms for the first time, a microbially driven Fe shuttle for the largest BIFs on Earth. In Project 3C: A redox-stratified ocean 3.2 billion years ago, a novel combination of stable Fe and radiogenic U–Th–Pb isotope data was used to demonstrate significant oxygen contents in the shallow oceans at 3.2 Ga, based on analysis of the Manzimnyama BIF, Fig Tree Group, South Africa. This unit is exceptional in that proximal, shallow-water and distal, deep-water facies are preserved. When compared to the distal, deep-water facies, the proximal samples show elevated U concentrations and moderately positive δ⁵⁶Fe values, indicating vertical stratification in dissolved oxygen contents. The relative enrichment of O₂ in the upper water column is likely due to the existence of oxygen-producing microorganisms such as cyanobacteria. These results provide a new approach for identifying free oxygen in Earth’s ancient oceans, including confirming the age of redox proxies, and indicate that cyanobacteria evolved prior to 3.2 Ga.

Two projects involved in situ stable isotope analysis via the SIMS at UW-Madison. In Project 3D: SIMS analyses of filamentous fossil microbes from the ~3,465 Ma-old Apex chert may reveal their physiology, Co-I’s Bill Schopf and John Valley began study of microfossils in the Apex chert. The 3.5 Ga Apex chert has been proposed to contain the oldest known microfossils, which early work suggested were analogous to cyanobacteria, and thus potentially oxygenic phototrophs. The biogenicity of these samples, however, has been questioned, but new SIMS isotope analysis of ¹³C/¹²C ratios of other microfossils suggests that correlation between morphology and isotopic compositions can distinguish abiologic from biologic sources. This, in combination with 3D laser Raman spectroscopy and 3D imaging, offers an opportunity to test divergent proposals for the origin of organic matter in the Apex chert. In addition, the specific isotopic compositions may help identify their photoautotrophic and/or methanotrophic physiology. In Project 3E: Genesis of high-δ¹⁸O Archean chert, Pilbara craton, Australia, Co-I John Valley led an effort to test approaches for inferring the temperature of the early Archean ocean through O isotope measurements of chert. Previous work on bulk samples identified moderately low δ¹⁸O values, which could indicate a very high temperature for the early Archean oceans. Alternatively, post-formation alteration may have changed the δ18O values. In the new work, detailed petrography of the 3.4 Ga Strelly Pool chert (Australia) was combined with in situ oxygen isotope analysis by SIMS. Values of δ¹⁸O vary from 10 to 30 ‰ within single outcrops. These results indicate that seawater temperatures of >70 oC that have been previously proposed for the early Archean are not supported.

In Project 3F: Searching for ancient impact events through detrital shocked zircons, Co-I Aaron Cavosie led a research effort that involved astrobiology summer intern students from the University of Puerto Rico. This group completed new surveys of detrital zircons from various localities, including a mixture of recent [Rock Elm (WI), Santa Fe (NM), Vredefort Dome (South Africa)], and ancient (Sudbury basin, Ontario) sediments. Detrital shocked zircons remain difficult to identify in Precambrian deposits, however studies of detrital shocked zircons in more recent sediments continue to provide extraordinary insights in areas as diverse as the distribution of high pressure phases, and ages of impacts on the Moon. The search for early Archean and Hadean age detrital zircons, which would provide insights into the very early Earth, and possibly evaluate the existence or nature of the “Late Heavy Bombardment”, continues.

Co-I Bill Schopf led Project 3G: A 3,400 Ma-old shallow water anaerobic sulfuretum evidences the anoxic Archean atmosphere, which documented fossilized sulfuretum bacteria in the 3.4 Ga Strelley Pool Chert, Australia. The anaerobic physiology of sulfuretum microbes indicates that Earth’s surface at this time was anoxic. Schopf suggests that the anaerobic H₂S-producing sulfuretum microbes may have coexisted with H2S-consuming anoxygenic phototrophs, lending support to the idea that the very old stromatolites in the Strelley Pool Chert may have been anoxygenic phototrophs.

Theme 4: Developing new Astrobiology Technologies

Two projects were pursued that were designed to develop new analytical approaches for studying astrobiological samples, including those that may be eventually returned from Mars. In Project 4A: New in situ techniques (CLSM and Raman) solve the problem presented by the disaggregation of acid-macerated organic-walled microfossils, Co-I Bill Schopf lead an effort to develop new methods for the non-destructive in situ analysis of organic carbon and organic-walled microfossils. This work uses confocal laser scanning microscopy (CLSM) and Raman spectroscopy. A test study was completed on bisaccate pollen from the Irati Subgroup (Permian, Paraná Basin, Brazil), and the results from these well-preserved specimens will be compared to samples of Precambrian age that are less well preserved. In Project 4B: New standards for analysis of O and C isotope ratios in Ca-Mg-Fe carbonates, led by Co-I John Valley, new standards were developed for SIMS analysis of O and C isotope ratios in Ca-Mg-Fe carbonates. Although carbonates are exceedingly important to understand because of the information they provide on biosignatures and paleoenvironments (see Theme 2 above), they are one of the most challenging minerals to analyze by SIMS due to exceptionally large compositional effects on instrumental mass bias. Over 70 different samples of dolomite to ankerite were examined by optical methods, SEM, EPMA, SIMS, and conventional stable isotope analysis. Thirteen samples possess the required homogeneity to be used as standards. The new calibration scheme shows that previous analyses are up to 5 ‰ in error due to non-linearity of the working curves, which would be equivalent to an error in estimated paleo temperature of ±30-35 oC, which encompasses most of the range proposed for the Precambrian oceans.