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Project Progress

1. Preservation of Molecular Biosignatures

Co-Investigator 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 polycyclic aromatic 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.

Fries and colleagues used the WiTec instrument for characterizing kerogens from a variety of samples. Murchison Fjord is a field site, on the northwest coast of Nordauslandet in the Svalbard archipelago bearing 750-800 Ma stromatolites and lagoonal carbonates. Columnar stromatolites from Murchison Fjord, Svalbard, bear horizontal carbonate layers of sub-mm thickness that alternate between fine-grained carbonate, carbonate with carbon-bearing detritus, and carbonates that bear distinct branching filamentous microfossils. Confocal Raman analysis is used to characterize microfossils in situ in terms of morphology and phase composition, including carbonaceous and sulfide species. Complementary whole-rock analysis of stable isotopes and molecular biomarkers are used to provide chemical context and constrain biological provenance. Analyses of stromatolitic microfossils from Murchison Fjord are being compared with similar structures found in other microfossils such as those found in the Apex, Strelley Pool, and Rhynie cherts, and the Tumbiana Formation.

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. Doctoral Student Rachel Schelble has carried out inorganic and organic carbon isotope analysis, mineralogic characterization, and scanning electron microscopy on microdrilled layers of core samples with the aim of identifying potential biosignatures that may be remnant in diagenetically altered manganese and iron ores from the ~2.0 Ga Mamatwan mine in the Kalahari Manganese Field, South Africa. Inorganic carbon isotopes in the ~43-ms thick lower manganese ore body showed a trend with more depleted δ¹³C_inorg at the initiation of manganese deposition and more enriched δ¹³C_inorg isotopes at the end of the sequence. The percentage of carbonate in the samples correlated with the δ¹³C of inorganic carbon isotopes in the lower manganese ore body. Manganese and iron-rich layers that contained relatively more wt% organic carbon were investigated for microfossils, but none were seen. On the basis of a bio-centric model of formation for the Kalahari Manganese Field, a transgression/regression model is consistent with the assumption that more depleted inorganic carbon isotopes represent closer proximity to an organic matter source.

Steele and Doctoral Student Maia Schweizer studied the exquisitely preserved Neoproterozoic animal embryos and other microfossils from the Doushantuo phosphorites in Wengan, Guizhou Province, China, using confocal Raman imaging (Figure 1). These microfossils represent Earth’s earliest multicellular life though their affinities remain a topic of debate, in part due to the lack of understanding of their taphonomy. A number of putative embryos at various division stages were examined in thin section. Confocal Raman imaging was used to investigate the quality of carbon preservation in morphological features of diagenetic and biological origins as well as the lithology of the host phosphorite. Most microfossils contain significant amounts of organic carbon in addition to phosphorite, carbonate, silica, and even clay minerals, which may have been deposited in or forced into the microfossils during diagenesis. Organic carbon in Doushantuo microfossils consists of both disordered and graphitic components (bands 1350 and 1580 cm^-1, respectively), which indicates a low level of thermal alteration. The matrix contains very little carbon. Membrane-like structures composed of phosphate and carbon, approximately 1 m in diameter, sometimes form invaginated layers at microfossil boundaries and, more rarely, occur within divided cells in a single embryo. Other microfossils exhibit zoning of phosphate and carbonate at their outer boundaries, possibly due to chemical alteration after early diagenesis. These compositional features shed light on the taphonomic processes responsible for the unusual preservation of Doushantuo microfossils as well as new biological features with possible taxonomic implications.

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Endolithic habitats are a common microbial adaptation to the inhospitable cold, dry arctic climate of Svalbard. Organisms take to fissures, cracks, and pores of rocks to escape the extremely harsh surface microenvironment. Both the arctic climate and endolithic habitat are analogs to Martian surface climate and predicted microbial habitat. Tests for patterns among genetic, lipid, and other high molecular weight compounds, and isotopic biomarkers collected during the 2005 AMASE expedition to Northern Svalbard were made by Postdoctoral Fellow Jennifer Eigenbrode and collaborators. In situ DNA sampling, PCR, and analysis performed in the field using gene-specific primers revealed the presence of cyanobacteria and fungi. PCR results also indicated differences in overall community composition and activity among different rock types. Similarly, preliminary lipid analyses delineate variations in cyanobacterial and eukaryote lipid signatures in fossil travertine, sandstone, and gypsum nodules containing endoliths. Using surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry, over 90 different compounds ranging in molecular weight from >500 to 2,500 daltons were found and will be correlated to microbial biomarkers. Carbon and nitrogen isotopic compositions will also be added to develop the characterization of endoliths. When successful, biosignature correlations strengthen interpretations and overcome interpretive ambiguities and may be useful in the search for life on Mars.

Former Postdoctoral Fellow Jan Toporski, Collaborator Jake Maule, and Steele studied volcanic environments long known to harbor microbial life of great diversity. This information was usually obtained from samples returned to laboratories, analysis of field sites for their microbiology in the field remained difficult. With planetary exploration missions dedicated to carry out in situ life detection experiments, the capability for assessment for microbial activity has gain increased relevance. Molecular tools including adenosine triphosphate (ATP) luminometry, lipopolysaccharide (LPS) detection, and antibody microarray technology have been deployed in volcanic environments of the Kamchatka peninsula, Russia. The specific field site is a young (< 2 years old) hydrothermal spring in the active crater of the Mutnowsky volcano. Initial results from ATP measurements of samples from a hot spring in this crater showed that there was no detectable microbial activity in the pool water (> 90°C). In contrast, high activity was found on the edges of the pool (~ 60°C). Portable Test System (PTS) measurements showed that high microbial activity corresponded well with high cell concentrations and vice versa. Microarray analysis of these samples confirmed this observation, as DNA was found to be present on the edge of the pool yet not in the pool water. These measurements further indicated the presence of melanoidins in the pool water, yet not at the edges. These studies, carried out on site, showed that microbial life is present and active at the edge of this young hydrothermal pool, and the breakdown products of microbes in form of melanoidins are recycled in the water of the pool.

Visiting Investigator Liane Benning (University of Leeds) worked with Steele and Fogel on a study of snow algae collected on the AMASE expedition (Figure 2). In their quest for survival in adverse terrestrial environments, microorganisms have developed a suite of protective compounds including scytonemin (UV-radiation screening pigment), phycocyanin (light-harvesting pigment), and carotene (antioxidant). In addition, in extremely cold environments such as the Arctic, microorganisms have developed strategies that allow them to thrive (in some cases more than 10⁶ cells per ml) under conditions of nutrient depletion, extreme temperature fluctuations, acidic conditions (pH 4.4 – 6.3), large osmotic variations during melting or ice formation, dehydration, and high levels of UV-irradiation. The group made Raman, microscopic/elemental, isotopic, and molecular microbiology measurements of snowfield algal communities from the Mars analog site in the Murchison Fjord area of Svalbard. The algae/microbe communities give the snow colors ranging from bright red through green, yellow, and orange to brown or even black. The main algal order responsible for the red and green colored snow is Chlamydomonadales (Figure 3). This algae developed secondary protective carotenoids (mainly astaxanthin) as well as resting cells and zygotes with thick cell walls or mucilaginous envelopes that act as protecting agents but also encourage the commonly observed surface coverage by mineral grains. In addition, algae of the genera Raphidonema, and Nostoc, some of which do not possess the pigment adaptations of Chlamydomonadales, as well as diatoms were present. The group’s results indicate the presence of about 5 different types of microalgae, all closely associated with a series of symbiotic microbial communities that impart a distinct community structure and that may affect the survival strategies of the microalgae.

Intern Rachel Erdil and Fogel used the ProteinChip reader to study the dissolved organic matter in ultra-basic springs at The Cedars in Sonoma County, California. The nature of the dissolved organic matter (DOM) present in ultra-basic springs in the highly serpentinized region of The Cedars in Sonoma County, California, was investigated using a Ciphergen Biosystems

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ProteinChip® Reader Surface Enhanced Laser Desorption/Ionization time-of-flight mass spectrometer (SELDI-TOF-MS). DOM, carbonate rock, and biofilm samples were obtained from five springs where pH ranged from 7.5 to 11.5. Prior to testing, bacterial cells were lysed using buffer containing 0.5% Triton X-100 detergent. Carbonate rock samples were dissolved using ultra-pure 6 N HCl then buffered to pH 5.5 (Figure 4). All samples were tested using Normal Phase 20 (NP-20) Protein Chips, which selectively bind proteins and other large organic molecules. Spectral data showed a dramatic difference in the molecular size of biofilm, rock, and DOM samples, from 2000 to 50000 Daltons for biofilms, 200 to 2000 Daltons for rocks, and 600 to 5000 for DOM. DOM from alkaline and neutral springs had different molecular weight distribution, possibly reflecting environmental impact. Peaks recorded from DOM samples were compared with possible combinations of fragments of peptidoglycan from bacterial cell walls for both gram-positive and gram-negative bacteria. DOM samples were found to be composed of 39% gram-negative bacteria and 52% gram-positive bacteria with 9% of the fragments unidentified. Non-native molecules dominated the composition of DOM across both gram-positive and gram-negative bacteria, which can be explained by the fact that much of DOM is highly degraded material. This study provides the basis for further investigation of the DOM in The Cedars to differentiate between abiotic and biotic sources.

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2. Stable Isotope Biosignatures

Co-Investigator Robert Hazen and colleagues completed a study of newly discovered sedimentary structures produced by ancient microbial mats in Early Archean sandstones of the 3.2-Ga Moodies Group, South Africa, which differ fundamentally in appearance and genesis from early Archean stromatolites and bacterial cell fossils preserved in chert. Wrinkle structures, desiccation cracks, and roll-up structures record the previous existence of microbial mats that effectively stabilized sediment on the earliest known siliciclastic tidal flats. In thin-section, the sedimentary structures reveal carpet-like fabrics characteristic of microbial mats. Negative δ¹³C isotope ratios of —20.1 to —21.5 ± 0.2 ‰ are consistent with a biological origin for the carbon preserved in filament-like microstructures. The biogenicity of the sedimentary structures in the Moodies Group is substantiated by comparative studies on identical mat-related features from similar tidal habitats throughout Earth history including the Recent. This study suggests that siliciclastic tidal flat settings have been the habitat of thriving microbial ecosystems for at least 3.2 billion years. Independent of controversial silicified microfossils and stromatolites, the newly detected microbially induced sedimentary structures in sandstone support the presence of bacterial life in the Early Archean.

Fogel began a collaboration with Daniel Glavin and Oliver Botta (NASA Goddard Space Flight Center) and Zita Martins (University of Leiden) on compound-specific isotope analysis in nucleobases isolated from the Murchison chondrite. Carbonaceous chondrites contain many biologically relevant organic molecules and may have delivered N-heterocycles including nucleobases to the Earth’s surface throughout history. It is possible that these extraterrestrial nucleobases were utilized and may have contributed to the origin of Earth’s first living systems. Confirmation of this hypothesis has been hindered by suspicion that meteoritic nucleobases may not be indigenous, but rather could be due to terrestrial contamination or are analytical artefacts. Carbon isotope data obtained by Fogel’s group indicate that at least one of the five nucleobases used in terrestrial nucleic acids, and one other structurally related compound, are indigenous components of the Murchison meteorite. A pyrimidine (uracil, δ¹³C +44.5‰) and a purine (xanthine, δ¹³C +37.7‰) both display non-terrestrial values. These data provide strong evidence that nucleobases, or their precursors, were delivered to the early Earth by meteorites.

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 N 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. Nitrogen may be one of the most diagnostic elements in the search for life on other planets. On Earth, it is an essential element needed for protein and nucleic acid structures. Often, N is a limiting nutrient for the productivity of living organisms. An understanding of how nitrogen might be distributed in a landscape that had extinct or very coldadapted, slow-growing extant organisms should be useful for detecting unknown life forms. Fogel’s group studied the biogeochemistry of the nitrogen cycle in Northern Svalbard, Norway, by examining pools of inorganic and organic nitrogen in soils, rocks, microbes, stream waters, and plants. Because of the very cold and dry weather there, volcanic soils contained very low amounts of total nitrogen, unless perturbed by plants, microbes, or animals. In weathered volcanic soils, reduced nitrogen concentrations were higher, and oxidized nitrogen concentrations lower. The opposite was found in a weathered Devonian redbed soil. Endolithic communities of microbes inhabited almost every surface of collected rock sample, and their δ¹⁵N indicated that biological nitrogen fixation could be an important source of this nutrient. Soils and plants influenced by animals had considerably enriched or depleted δ¹⁵N from the average. Once living organisms had influenced a rock, it retained higher %N and the distinctive δ¹⁵N associated with biological production. Extreme cold slows biological rates and nitrogen cycling, such that analogues of cold environments have a mosaic of N pattersn, not at all expected without significant biological activity.

Co-Investigator Rumble and his colleagues measured oxygen isotopes in minerals that may contain tell-tale proxies for fractionation processes on δ¹⁷O — δ¹⁸O plots from deposits of rocks and minerals on the Earth or other planetary bodies. Kinetically-controlled versus equilibrium biochemical and geochemical processes may yield chemically-specific isotope fractionation such as that observed in the three-body ozone formation reaction. Rumble and colleagues tested whether the precision and accuracy of laser fluorination is capable of distinguishing small differences in slope by analyzing aliquots of the same mineral samples in two different laboratories. Laser fluorination analyses of aliquots of two groups of minerals for ¹⁶O-¹⁷O-¹⁸O show good agreement in measured values of the slopes of linearized δ¹⁷O vs. δ¹⁸O data.

While geochemical indicators or biosignatures, such as stable carbon and hydrogen isotope values, of biogenic hydrocarbons have been identified, less is known about unambiguous abiogenic hydrocarbons. Traditionally, hydrocarbons that are depleted in ¹³C are considered to be of biogenic origin, because many biological enzymatic reactions are known to preferentially incorporate the lighter carbon isotope. In a study by Postdoctoral Fellow Penny Morrill, laboratory experiments were designed to determine if a similar carbon isotopic depletion would be observed during abiogenic synthesis of hydrocarbons by a Fischer-Tropsch Type reaction. Carbon dioxide was reduced in the presence of a metal catalyst under hydrothermal conditions in sealed gold capsules. Hydrocarbon chains up to five carbons long were produced. These gases had relatively large carbon isotopic depletions, which mimic that of gases with biogenic origins. Compound-specific hydrogen isotopes measurements were preformed for the first time during experimental abiogenic formation of hydrocarbons (Figure 5). A hydrogen isotopic enrichment trend was observed with each additional carbon. Similar hydrogen isotopic enrichment was also seen in Canadian Shield hydrocarbons interpreted as abiogenic. This experimental data further emphases the need for both carbon and hydrogen isotope values to be used as key parameters for distinguishing biogenic from abiogenic gases.

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To investigate the possible abiogenic origin of hydrocarbons at a site of active serpentinization, Morrill performed compositional and isotopic analyses on gases collected from ultrabasic reducing springs flowing through serpentine and peridotite rocks in northern California. The sampled gases were primarily composed of nitrogen (≤ 72%), hydrogen (≤ 50%), and methane (≤ 17%). Other minor gases included argon and higher molecular weight hydrocarbons. The carbon and hydrogen isotope values of methane, with mean values of —64 and -335 ‰, respectively, indicate a biological source, namely, microbial methanogenesis (Figure 6). Higher hydrocarbons can be formed by thermal decomposition of larger organic molecules, or through abiotic polymerization of smaller organic molecules. C₂ – C₆ gases sampled at the site did not exhibit the pattern of ¹³C-enrichment with an increase in carbon number typically associated with the thermal degradation of organic matter. Instead, these compounds exhibited altering ¹³C-enrichment and depletion with each additional carbon. This pattern was also observed in Canadian Shield hydrocarbons interpreted as abiogenic. Therefore, these gases from the ultrabasic reducing springs resemble gases of an abiogenic origin. Higher molecular weight hydrocarbon gases (straight chain and cyclo-alkanes) were also found with a mean carbon isotope value of -25‰. This third group of gases has similar compositions and isotope values to thermogenic gases suggesting they may be migrated products of thermal degradation of buried organic material. In summary, this study demonstrates how compositional and isotopic analyses can help to identify pathways (biotic and abiotic) for hydrocarbon gas formation.

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The distribution of ¹³C/¹²C isotope ratios in upper mantle ultramafic xenoliths exhibits a distinct bimodality with one δ¹³C maximum near -25 ‰ and another near -5 ‰. A similar bimodality has been observed in the Bockfjorden Volcanic Complex (BVC) lherzolite xenoliths from northwest Spitsbergen. The light isotope fraction commonly has been suggested as due to an organic, low-temperature component. However, there are data indicating that a portion of this fraction is retained in upper mantle silicates from stepwise heating above 1350˚C. Interestingly, in a preliminary experiment with diopside+H₂O+CO₂ at 1.5 GPa and 1150˚C, Morrill and colleagues found a ¹³C/¹²C fractionation between -10 and -20 ‰. The suggested large fractionation between carbon-bearing gas and silicate minerals at upper mantle pressures and temperatures is qualitatively consistent with calculated fractionation factors between gas in the C-O-H system and CaCO₃ and elemental carbon. In addition to temperature, the fractionation factor is sensitive to the gas species (oxidation state of carbon).

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

During the past year Co-Investigator Cody and colleagues have been collaborating with a number of groups through the application of molecular spectroscopic analysis of ancient and modern complex biochemical solids. The methods employed are solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray absorption spectroscopy (e.g., carbon, nitrogen, and oxygen X-ray absorption near edge spectroscopy, XANES). Cody and colleagues have continued their ongoing collaboration with the UCLA NAI team through the analysis of ancient (50 Ma) fossil ferns and a comparative study of laboratory-heated modern ferns to investigate the pathways by which fossil organic matter is transformed and ultimately preserved. This project involves primarily Cody and Andy Czaja and William Schopf of UCLA. Cody and colleagues are also using solid-state ¹³C NMR to characterize microbial biofilms in collaboration with Jill Banfield and her group in the Berkeley NAI team. In particular, they are attempting to verify the presence of specific extra-cellular polysaccharides predicted via proteomics to be present in the biofilms. Finally, Cody and colleagues are applying C-, N-, and O-XANES to explore the chemistry of Archean kerogens from the Hammersley Basin, Australia. These kerogens are up to 2.7 Ga in age and, although they have been subjected to significant geochemical transformation, retain considerable carbon, nitrogen, and oxygen functional group information that signifies their biological origins. These studies are essential for subsequent studies of even more ancient kerogens, whose origins are controversial from the standpoint of biological or abiotic processes.