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Project Reports

• Developing New Biosignatures

  The development and experimental testing of potential indicators of life is essential for providing a critical scientific basis for the exploration of life in the cosmos. In microbial cultures, potential new biosignatures can be found among isotopic ratios, elemental compositions, and chemical changes to the growth media. Additionally, life can be detected and investigated in natural systems by directing cutting-edge instrumentation towards the investigation of microbial cells, microbial fossils, and microbial geochemical products. This project combines our geomicrobiological expertise and on-going field-based environmental investigations with a new generation of instruments capable of revealing diagnostic biosignatures. Our efforts are focused on creating innovative approaches for the analyses of cells and other organic material, finding ways in which metal abundances and isotope systems reflect life, and developing creative approaches for using environmental DNA to study present and past life.

  Apatite-biomineralized Neoproterozoic protists Using specimens provided by N. J. Butterfield (Cambridge University) and P.A. Cohen (Harvard University), our studies by optical microscopy, confocal laser scanning electron microscopy, and Raman spectroscopy show that ~775-Ma-old protistan scale fossils (from Yukon Territory, Canada) were originally composed of apatite — a surprising discovery, given that the scales of extant protists are most commonly silica or calcite. The Yukon Territory fossils, the earliest example of biomineralization now known from the geological record, have recently been reported by our group in Geology Today; a more detailed manuscript reporting these findings was published in Geology.

  Chert-permineralized Paleoproterozoic sulfuretum

  Using samples originally provided by Martin Van Kranendonk, augmented by samples that Malcolm Walter and I collected in June 2011, we have discovered a ~2,300-Ma-old microbial ecosystem previously unknown in Precambrian deposits. This ecosystem, composed of sulfate-reducing and sulfur-oxidizing bacteria, is remarkably similar to extant sulfureta first described by Victor Gallardo and Carola Espinoza in 2007 from numerous core samples collected at the sediment-water interface in the sub-oxygen minimum zone off the western coast of South America. The stratigraphy and geologic setting indicates that the fossiliferous rocks were deposited at depth, well below wave base and most likely below the photic zone. Geochemical data are consistent with the presence of sulfate reducers. The major taxa of the abundant, diverse assemblage are morphologically distinct from fossil and modern cyanobacteria but are essentially identical to sulfur-oxidizing bacteria described by Gallardo and Espinoza. Similarly, the wispy cobweb fabric of the microbial assemblage differs distinctly from the laminated fabric of photoautotroph-dominated stromatolite/microbial mat communities but is indistinguishable from the fabric of modern sulfureta. When current SIMS work is completed, designed to measure the carbon isotopic composition of individual benthic and planktonic members of the assemblage (and based on the pioneering work of Christopher House, published in 2000), a paper reporting these new findings will be submitted to Nature.

  Our studies of extant microorganisms (biology), fossil megascopic and microscopic organisms (paleontology), the stratigraphy, mineralogy and taphonomy of their preservation (geology), and their chemical and isotopic composition (geochemistry) are highly interdisciplinary, involving both the life sciences (biology, microbiology, evolutionary biology) and the physical sciences (geology and geochemistry).

  In addition to the UCLA contributors noted above (J. William Schopf, Anatoliy B. Kudryavtsev, and Ian B. Foster), contributors to this research include: Jack D. Farmer (NAI Astrobiology Center, Arizona State University); Malcolm R. Walter and Martin Van Kranendonk (Australian Centre for Astrobiology, University of New South Wales, Sydney, Australia); Kliti Grice (Curtin University, Perth, Australia); John Valley and Ken Williford (NAI Astrobiology Center, University of Wisconsin); Victor Gallardo and Carola Espinoza (Universidad de Concepción, Chile); Phoebe A. Cohen (MIT); Francis Macdonald (Harvard University); and Nicolas J. Butterfield (Cambridge University)

  Subproject: Weathering and inorganic chemical biosignatures

  Secondary minerals and regolith profiles on basaltic rocks

  Based upon images taken during the 2003-2004 series of missions to Mars, several different weathering features appear to be present on the Mars surface including surface flaking, dirt cracking, exfoliation, honeycomb weathering and the formation of what appear to be weathering rinds (Thomas et al., 2005). However, the underlying mechanisms responsible for these features are difficult to ascertain from images alone. The goal of this study is to understand the role that different climatic conditions on Earth play in facilitating the weathering of basalts, which are thought to be the predominant lithology present on Mars. Basalts from three different sites are compared, two of which come from the temperate northeastern United States, while the third site is located in the dry, Arctic region of Svalbard and is thus considered to be a Mars analogue. Soil profiles from the two North American sites are much deeper than those from Svalbard and show clear evidence of chemical weathering in the bulk soil profile. While no evidence for chemical weathering is observed in bulk soils from Svalbard, the clay-sized fraction nevertheless shows clear evidence for the elemental depletion of K, Na, Ca and Mg. In contrast, Al and Fe appear to be immobile even in the finest size fractions of the Svalbard soil. Coupled with SEM images showing evidence of spalling on the outermost 100 μm of Svalbard rocks, this chemical evidence suggests that the process of spalling in cold climates may preferentially remove certain elements over others both on Earth as well as on Mars. Extractions with 0.5 N HCl, designed operationally to remove amorphous Fe oxides, indicate that the prevalence of amorphous Fe is greatest in the lowermost horizons of a Pennsylvania diabase, a finding that has been noted previously in a granodiorite-derived soil profile from Puerto Rico that is thought to contain a lithoautotrophic Fe-oxidizing community at depth (Buss et al., 2010). In further agreement with findings from Puerto Rico, Fe(II) concentrations in the lowermost soil of the Pennsylvania site decrease over a short distance as a results of rapid oxidation in the lowermost portion of the 4-m profile analyzed here. In contrast, no such change in extractable Fe concentrations or Fe(II) content is noted within the regolith of the Svalbard Mars analogue site, at which Fe-oxidizing bacteria are presumably absent.

  DNA has been extracted from several of the sampling sites on Svalbard, and metagenomic sequencing carried out. Preliminary analysis of the sequence data shows that while Proteobacteria, Actinobacteria, Acidobacteria, and Bacteroidetes are the dominant phyla at all sites sequenced, there are significant differences in phylum relative abundance and community structure between sites.

  Community structure and geochemistry of weathering in a Puerto Rican watershed (Note: Primary funding for this project is through DOE)

  We have hypothesized that Fe oxidation may be important in weathering of intact bedrock to disaggregated regolith at depth (9.2 m) in the Bisley volcaniclastic watershed in the Luquillo Mountains of Puerto Rico. The regolith forms along a gradient between conditions of low oxygen and higher pH at depth, to higher oxygen and lower-pH waters at the surface. The active weathering front was observed to be ~ 8.3 m, as secondary clay minerals and Fe oxides are present from the surface down to this depth, while the primary minerals chlorite and feldspars were detected below 8.3 m. Consistent with an active weathering front at ~ 8.3 m, ammonium-acetate extracted Ca and Mg were present in higher concentrations below 7 m. There were also increases in both total and heterotrophic cell counts in this region, as well as increased concentrations of HCl-extracted Fe(II) and organic-bound Fe. As a consequence of these gradients in oxygen, pH, and chemical composition, we expect to see changes in microbial communities along the regolith. Metagenomics analysis revealed that sub-phylum groups containing known iron-oxidizing microorganisms were found in the deepest samples, near the regolith-bedrock interface, consistent with the hypothesis that chemolithoautotrophic bacteria play an important role in weathering and element cycling in the deep regolith. Rarefaction curves generated at a 97% similarity value indicated that the regolith was representatively sampled for determining community richness.

  Subproject: Intermolecular Isotopic Patterns

  F430 Isolation and isotopic characterization

  Methyl-coenzyme M reductase, an enzyme that plays a key role in methanogenesis, has recently been linked also to the anaerobic oxidation of methane (Scheller, 2010). Cofactor F430 is a tetrapyrrole nickel complex contained within the active site of methyl-coenzyme M and appears to be used in both methanogenesis and methanotrophy (Scheller, 2010; Mayer et al., 2008). Our efforts have been focused on purifying F430 from natural samples in order to determine δ¹⁵N and δ¹³C values both at natural abundance and in isotopically enriched samples from tracer studies. F430 is isolated using multidimensional preparatory and high-performance chromatographic separations. Compound identity and purity are confirmed using molar C:N ratios, light absorbance and MSⁿ detection and fragmentation of F430 isolated from pure cultures of Methanosarcina acetivorans. Our efforts to measure C isotope abundances using nano-EA/IRMS (Polissar et al., 2009) were challenged by the presence of co-eluting non-tetrapyrrole structures, and this has been addressed through refined HPLC separation on a hydrocarb column. Nitrogen isotopic measurements have been successful on standards and natural samples. F430 increases in abundance in environmental samples with active anaerobic methane oxidation in a low oxygen environment and provides support for an oxidation pathway that includes reversal of the enzymes involved in methanogenesis.

  Tininoid Isotopic Analysis: Pilot Study

  Tanja Bosak and her coworkers (Bosak et al., 2011) have recently documented well preserved microfossils in limestrones of Mongolia at 715-638 Ma. These small (~100 μm) organic-walled fossils morphologically resemble tintinnids, modern planktonic ciliates. Bosak has identified these unique microfossils in Cryogenian strata associated with the Tayshir isotopic excursion in carbonate and organic carbon. A new collaboration between Penn State and MIT researchers aims to perform isotopic analyses of these microfossils in order to provide insights to potential role(s) of tintinnoids in the carbon cycle of the ancient ocean. To date, test analyses have been conducted for pilot samples and reveal δ¹³C values that are consistent with those of modern planktonic organisms. Nitrogen isotopic abundances are slightly enriched relative to values in modern marine eukaryotes. Work is underway to scale up analyses from this pilot study and evaluate across the Mongolian section

  Scytonemin: a biomarker for cyanobacterial in harsh environments

  We have advanced our study of scytonemin, including new stable isotopic data to identify provenance, and strengthening interpretations based on its occurrence in ancient sediments. Scytonemin is found in extracellular polysaccharide sheaths of terrestrial or benthic cyanobacteria and is most abundant when cells are exposed to direct sunlight, such as in desert soil crusts and intertidal mats. Scytonemin is preserved in abundance in mid-Holocene sedimentary intervals in the Black Sea, a novel deep-sea occurrence that demonstrates that scytonemin may be preserved in other marine or lacustrine sapropel sediments. We report new results of both C and N isotopic compositions from modern settings and for the sedimentary compounds and these findings support the interpretation that scytonemin was derived from cyanobacteria in cryptobiotic desert soils, resisting degradation during erosion and transport. Its occurrence points toward the expansion of arid conditions during the Subboreal Phase (5.5-2 ka, during Unit IIa deposition) in the Black Sea region. Because it survives under harsh irradiative and oxidative conditions and during terrestrial transport processes, scytonemin has potential to serve as an important and diagnostic organic biomarker for tracing the evolution and expansion of cyanobacterial populations especially in association with elevated UV stress.

  Subproject: Abiotic production of organic material

  In collaboration with the NAI team at NASA Goddard, we have been looking at both pre-RNA chemistry and organics in meteorites. Graduate student Karen Smith published with the NASA Goddard group a paper in PNAS on purine diversity in primitive meteorites. The work shows non-biological purines in meteorites, and also reveals that the abundance of purines in meteorites is likely related to aqueous alteration. Karen is continuing work on the organics in meterorites, as well as her work on pre-RNA chemistry. Undergraduate student Danielle Gruen is continuing studies of HCN production under different atmospheric conditions.

  Subproject: Using Methanosarcina to develop new biosignatures

  Long chain poly-P is ubiquitous to microbial life on Earth (Proc. Natl. Acad. Sci. USA (2004) 101:16085), although few investigations have been reported in species from the Archaea domain. A role for poly-P in stress response has been suggested for the methane-producing archaeon Methanosarcina acetivorans (J. Proteome Res. (2007) 6:759). The probable ancient origin and widespread occurrence in diverse microbes, coupled with the role in coping with extreme environments, identifies poly-P as a facile biosignature for exploration of extraterrestrial life. We are developing poly-P for use as a biosignature by investigating poly-P metabolism in the domain Archaea with M. acetivorans for which the genome is annotated with genes encoding two putative poly-P kinases (Ppk1 and Ppk2) catalyzing synthesis of poly-P.

  The levels of polyphosphate (poly-P) during methanol- or acetate-dependent growth of Methanosarcina acetivorans were measured to begin to assess the physiological role. The results (Fig. 1) show that levels increase with growth and then rapidly decline at the onset of stationary phase in methanol-grown cells, although well before stationary phase in acetate-grown cells suggesting different roles for poly-P depending on the growth substrate. Furthermore, the maximum level of poly-P accumulation at any time during growth with acetate was substantially lower compared to growth with methanol.

  Beta-glucuronidase:polyphosphate kinase reporter gene fusions were constructed to assess expression of the kinase during growth with methanol vs. acetate. The results in Figure 4 show greater levels of expression during growth with acetate vs. methanol. The most parsimonious interpretation is that the lower levels of poly-P in acetate-grown cells are the consequence of increased utilization rather than synthesis indicating an important role for poly-P during growth with acetate.

  Subproject: Developing Secondary Ion Mass Spectrometry (SIMS) as a tool for Astrobiology

  During the past year, we started to develop new samples and standards for the analysis of ancient microfossils and organic material using SIMS.

  Subproject: Isotopic biosignatures in minerals

  The generation and recording of isotopic biosignatures in minerals is the general focus of this subproject. We are specifically interested in determining if microbes interact with their surrounding environment in a manner that affects the isotopic composition of minerals. We begin this investigation in year 1 by collecting gypsum samples from sulfidic caves and measuring the calcium and sulfur isotopic composition of these samples. In year 2, we initiated abiotic experiments aimed at elucidating the abiotic isotope effects, thus serving as context for evaluating the previous measurements.

  In year 3, we completed two main tasks. One, we completed the initial Ca isotope experiments and measured the Ca isotopic composition of the experiments at IFM-GEOMAR in Kiel, GER (Figure 5). This work is the foundation of Harouaka’s Masters thesis. She will graduate in December 2011 and plans to continue her studies in astrobiology working towards a PhD at PSU. Two, Fantle, Harouaka, and Gonzales traveled to the Frasassi caves and sampled atmospheric sulfide for S isotopic analysis and additional gypsum from the cave walls at Grotta Bella in order to characterize the system better.

  We also initiated two preliminary studies with the help of undergraduate researchers. The first was a study of low temperature anhydrite formation, which was the basis of Present’s senior thesis. The second is an on-going study of laboratory cultures of A. thiooxidans, work that Mansor is conducting as an UGA. Manor is coadvised by Fantle and Macalady. The latter study is helping us characterize A. thiooxidans morphologically and geochemically and we hope to use this information to design isotope experiments over the next year.

  Subproject: DNA as a biosignature

  Ancient DNA and DNA preservation

  Dr. Beth Shapiro (PSU) collected a series of new permafrost cores during the last field season, from which experiments about the rate and process of DNA decay is now underway. Her team is implementing a new drilling technique to spike samples with bacteria whose genomes contain the pET23a-gfp construct at the time of drilling. This enables extraordinary sensitivity with regard to permafrost thaw, DNA migration and contamination. In addition, this provides a means to monitor and evaluate DNA decay via irradiation. In addition, her work with fossil DNA has resulted in the publication of six peer-reviewed manuscripts, with five more in press.

  Metagenomics as a tool for Astrobiology

  The House research group participated in two IODP expeditions to gain new samples for the developing of metagenomics for Astrobiology. Leah Brandt and Chris House were shipboard participants in IODP Expedition 331 to study the Deep Hot Biosphere of the Okinawa Hydrothermal Mounds. Then, Mandi Martino was the shipboard microbiologist for IODP Expedition 334 to the Costa Rica margin. These new exciting locations will be very useful for our present work. We also published a paper on the metagenomics of the subsurface Brazos-Trinity Basin (IODP Site 1320), which provided a initial view of the subsurface microbiology at a sandy, low biomass continental margin site. Finally, we have submitted a paper on our newly-developed method for amplifying DNA from low biomass environmental samples. This paper provides a useful method for amplification when the work requires good negative blanks, often essential for applying metagenomics in astrobiology.

  This reporting period say the initiation of our Citizen Science Project on thermophiles in domestic water heaters. During the reporting period, we developed the kits that will be sent to participants and recruited participants from across the United States. The kits will be sent out Fall 2011 and returned to our labs in early 2012. Additionally, we isolated a novel Halomonas from wastewater which can grow at a remarkable range of pH and salinity. It is also highly resistant to Asenate. Finally, we also initiated a new collaboration with astrobiologists in Colombian.

  ROADMAP OBJECTIVES: 5.1

• AIRFrame Technical Infrastructure and Visualization Software Evaluation

  We have analyzed over four thousand astrobiology articles from the scientific press, published over ten years to search for clues about their underlying connections. This information can be used to build tools and technologies that guide scientists quickly across vast, interdisciplinary libraries towards the diverse works of most relevance to them.

  ROADMAP OBJECTIVES: 1.1 1.2 2.1 2.2 3.1 3.2 3.3 3.4 4.1 4.2 4.3 5.1 5.2 5.3 6.1 6.2 7.1 7.2

• Advancing Methods for the Analyses of Organics Molecules in Sediments

  Eigenbrode’s astrobiological research focuses on understanding the formation and preservation of organic and isotopic sedimentary records of ancient Earth, Mars, and icy bodies. To this end, and as part of GCA’s Theme IV effort, Eigenbrode seeks to overcome sampling and analytical challenges associated with organic analyses of astrobiology relevant samples with modification and development of contamination tracking, sampling, and analytical methods (primarily GCMS) that improve the recovery of meaningful observations and provide protocol guidance for future astrobiological missions. Advances have been made in five sub-studies and manuscript writing is in progress. Studies include: 1 & 2. Advancing protocols for organic molecular studies of iron-oxide rich sediments and sediments laden with perchlorate, 3. Carbon Isotopic Records of the Neoarchean, 4. Solid-phase sorbtive extraction of organic molecules in glacial ice, and 5. Amino acid composition of glacial ice.

  ROADMAP OBJECTIVES: 2.1 4.1 5.1 5.2 5.3 6.1

• Biosignatures in Relevant Microbial Ecosystems

  In this project, PSARC team members explore the isotope ratios, gene sequences, minerals, organic molecules, and other signatures of life in modern environments that have important similarities with early earth conditions, or with life that may be present elsewhere in the solar system and beyond. Many of these environments are “extreme” by human standards and/or have conditions that are at the limit for microbial life on Earth.

  ROADMAP OBJECTIVES: 4.1 4.3 5.1 5.2 5.3 6.1 7.1 7.2

• Habitability of Icy Worlds

  Habitability of Icy Worlds investigates the habitability of liquid water environments in icy worlds, with a focus on what processes may give rise to life, what processes may sustain life, and what processes may deliver that life to the surface. Habitability of Icy Worlds investigation has three major objectives. Objective 1, Seafloor Processes, explores conditions that might be conducive to originating and supporting life in icy world interiors. Objective 2, Ocean Processes, investigates the formation of prebiotic cell membranes under simulated deep-ocean conditions, and Objective 3, Ice Shell Processes, investigates astrobiological aspects of ice shell evolution.

  ROADMAP OBJECTIVES: 1.1 2.1 2.2 3.1 3.2 3.3 3.4 4.1 5.1 5.3 6.1 6.2 7.1 7.2

• Ecology of Extreme Environments: Characterization of Energy Flow, Bioenergetics, and Biodiversity in Early Earth Analog Ecosystems

  The distribution of organisms and their metabolic functions on Earth is rooted, at least in part, to the numerous adaptive radiations that have resulted in the ability to occupy new ecological niches through evolutionary time. Such responses are recorded in extant organismal geographic distribution patterns (e.g., habitat range), as well as in the genetic record of organisms. The extreme variation in the geochemical composition of present day hydrothermal environments is likely to encompass many of those that were present on early Earth, when key metabolic processes are thought to have evolved. Environments such Yellowstone National Park (YNP), Wyoming harbor >12,000 geothermal features that vary widely in temperature and geochemical composition. Such environments provide a field laboratory for examining the tendency for guilds of organisms to inhabit particular ecological niches and to define the range of geochemical conditions tolerated by that functional guild (i.e., habitat range or zone of habitability). In this aim, we are examining the distribution and diversity of genes that encode for target metalloproteins in YNP environments that harbor geochemical properties that are thought to be similar to those that characterize early Earth. Using a number of newly developed computational approaches, we have been able to deduce the primary environmental parameters that constrain the distribution of a number of functional processes and which underpin their diversity. Such information is central to constraining the parameter space of environment types that are likely to have facilitated the emergence of these metal-based biocatalysts.

  ROADMAP OBJECTIVES: 3.2 3.3 3.4 4.1 4.2 5.1 5.2 5.3

• Biosignatures in Ancient Rocks

  The Earth’s Archean and Proterozoic eons offer the best opportunity for investigating a microbial world, such as might be found elsewhere in the cosmos. The ancient record on Earth provides an opportunity to see what geochemical signatures are produced by microbial life and how these signatures are preserved over geologic time. As part of our integrated plan, we will study geochemical, isotopic, and sedimentary signatures of life in order to understand the context in which these biosignatures formed.

  ROADMAP OBJECTIVES: 1.1 3.2 4.1 4.2 4.3 5.1 5.2 5.3 6.1 6.2 7.1 7.2

• Survivability of Icy Worlds

  Investigation 2 focuses on survivability. As part of our Survivability investigation, we examine the similarities and differences between the abiotic chemistry of planetary ices irradiated with ultraviolet photons (UV), electrons, and ions, and the chemistry of biomolecules exposed to similar conditions. Can the chemical products resulting from these two scenarios be distinguished? Can viable microbes persist after exposure to such conditions? These are motivating questions for our investigation.

  ROADMAP OBJECTIVES: 2.2 3.2 5.1 5.3 7.1 7.2

• Molecular Evolution: A Top Down Approach to Examine the Origin of Key Biochemical Processes

  The emergence of metalloenzymes capable of activating substrates such as CO, N2, and H2, were significant advancements in biochemical reactivity and in the evolution of complex life. Examples of such enzymes include [FeFe]- and [NiFe]-hydrogenase that function in H2 metabolism, Mo-, V-, and Fe-nitrogenases that function in N2 reduction, and CO dehydrogenases that function in the oxidation of CO. Many of these metalloenzymes have closely related paralogs that catalyze distinctly different chemistries, an example being nitrogenase and its closely related paralog protochlorophyllide reductase that functions in the biosynthesis of bacteriochlorophyll (photosynthesis). While the amino acid composition of these closely related paralogs are often quite similar, their biochemical reactivity and substrate specificity are often very different. This phenomenon is a direct consequence of the composition and molecular structure of the active site metallocluster, which requires a number of accessory proteins to synthesize. By specifically focusing on the origin and subsequent evolution of these metallocluster biosynthesis proteins in relation to paralogous proteins that have left clear evidence in the geological record (photosynthesis and the rise of O2), we have been able to obtain significant insight into the origin and evolution of these functional processes, and to place these events in evolutionary time.

  The genomes of extant organisms provide detailed histories of key events in the evolution of complex biological processes such as CO, N2, and H2 metabolism. Advances in sequencing technology continue to increase the pace by which unique (meta)genomic data is being generated. This now makes it possible to seamlessly integrate genomic information into an evolutionary context and evaluate key events in the evolution of biological processes (e.g., gene duplications, fusions, and recruitments) within an Earth history framework. Here we describe progress in using such approaches in examining the evolution of CO, N2, and H2 metabolism.

  ROADMAP OBJECTIVES: 3.2 4.1 5.1

• Project 5: Geological-Biological Interactions

  This project involves multiple researchers exploring life in extreme environments, the signature of life (chemical, isotopic and mineralogical), and the adaption of life. All together the many sub-topics of this project seek to inform us about where to search for life on other worlds and how to seek evidence that life once existed on other worlds.

  ROADMAP OBJECTIVES: 4.1 5.1 6.1 6.2 7.1

• Project 2B: Proto-Cell Membrane Evolution May Have Been Directed by Mineral Surface Properties

  Metal oxides have been studied widely in the biogeochemical literature for understanding the adsorption and other surface interactions of dissolved organic and inorganic molecules with mineral surfaces. The goal of our study is to understand whether the earliest lipid membranes or “protocells” would have been stable in contact with different mineral surfaces on early Earth, and whether the surface properties of the minerals control their relative affinity to cell membranes. In previous years of this study, we used bulk adsorption isotherms and classical DLVO theory modeling approaches to examine the stability lipid bilayers in contact with micron-sized quartz (α-SiO2), rutile (α-TiO2) and corundum (α-Al2O3) particles. By understanding the role of natural geochemical parameters such as mineral surface chemistry, solution chemistry and temperature cycling on protocell membrane stability, we attempted to model potential aqueous environments where life may have originated such as lacustrine, tidal pool, and sub-aerial or submarine hydrothermal vents. In the present project year, we used neutron reflectivity to determine if the geometry of the mineral surface (sub-spherical particles versus planar single crystal surfaces) affects membrane stability. The results of our various approaches were consistent showing that lipid membrane stability depends on (1) lipid head-group charge and (2) surface charge of the mineral, which in turn depend on pH, ionic strength, presence or absence of Ca2+, (3) van der Waals interactions, and (4) relative hydrophobicity of the surface, as well as purely physical parameters such as relative size of the model membrane relative to the mineral surface. Our project addresses NASA Astrobiology Institute’s (NAI) Roadmap goals of understanding the origins of cellularity and the evolution of mechanisms for survival at environmental limits, and NASA’s Strategic Goal of advancing scientific knowledge of the origin and evolution of the Earth’s biosphere and the potential for life elsewhere.

  Keywords: lipid, protocell, vesicle, self-assembly, pre-biotic, mineral surface, hydrophilic, hydrophobic, bilayer

  ROADMAP OBJECTIVES: 3.4 5.1

• Evolution of Metabolism

  Our astrobiology research focus for VPL is to understand the evolution of different metabolic groups of microorganisms during the course of Earth’s history, and how the emergence of different metabolisms, such as methanogenesis, anoxic and oxygenic photosynthesis, and other anaerobic metabolisms that involve sulfur, metal, and nitrogen could effect the chemical composition of the atmosphere.

  ROADMAP OBJECTIVES: 5.1 5.3 6.2

• Metabolic Networks From Single Cells to Ecosystems

  Members of the Segre’ group use systems biology approaches to study the complex network of metabolic reactions that allow microbial cells to survive and reproduce under varying environmental conditions. The resource allocation problem that underlies these fundamental processes changes dramatically when multiple cells can compete or cooperate with each other, for example through metabolic cross-feeding. Through mathematical models of microbial ecosystems and computer simulations of spatially structured cell populations, the Segre’ team aims at understanding the environmental conditions and evolutionary processes that favor the emergence of multicellular organization in living systems.

  ROADMAP OBJECTIVES: 4.1 4.2 5.1 5.2 6.1

• Project 2C: Role of Extracellular Polymeric Substances (EPS) and Bacteria Cell Wall Structure in Shielding Against Specific Mineral Toxicity – Implications for Cell Surface Evolution

  Our interdisciplinary project examined the hypotheses that (1) bacterial cell membranes are ruptured in contact with specific mineral surfaces, (2) biofilm-forming extra-cellular polymeric substances (EPS) may have evolved to shield against membrane rupture (cell lysis), (3) differences in cell-wall structure of Gram-negative and Gram-positive bacteria may influence the susceptibility of cells to toxic minerals and (4) mineral toxicity depends on its surface chemistry and nanoparticle size.

  Previously, we have examined Gram-negative P. aeruginosa strains, wild-type (PAO1) that is capable of generating copious amount of EPS and producing biofilms, as well as the knock-out mutant (Δ-psl) that is defective in its ability to form EPS and biofilms. In the 2010-2011 year of funding, we have expanded our study to include Gram-positive B. subtilis strains, biofilm-producing wild-type NCIB3610 and biofilm-defective mutant yhxBΔ. We confirmed the hypotheses (1), (2) and (4) for both Gram-negative and Gram-positive bacteria, with toxicity increasing as amorphous β-TiO2 < γ-Al2O3. We also confirmed hypothesis (3) that Gram-positive bacteria are less susceptible to mineral toxicity than Gram-negative because of the most robust cell-wall structure of the former. Finally, we have shown that the mechanisms of toxicity depends on mineral surface charge for initial adhesion of nanoparticles to the cell surface, nanoparticle size which determines whether the particles can enter the intracellular space (e.g., for γ-Al2O3), the presence of surface free radicals (e.g., β-TiO2 ) which would have been generated by UV-radiation and meteorite impacts on early Earth, Mars, and other worlds.

  By understanding the mechanisms for membranolysis, especially under the extreme conditions of high radiation and heavy impacts during early planetary history, the project addresses the NASA Astrobiology Institute’s (NAI) Roadmap goals of understanding the origins of cellularity, the evolution of mechanisms for survival at environmental limits, and preservation of biosignatures, and NASA’s Strategic Goal of advancing scientific knowledge of the origin and evolution of the Earth’s biosphere and the potential for life elsewhere.

  ROADMAP OBJECTIVES: 3.4 5.1 7.1

• Postdoctoral Fellow Report: Steven Mielke

  This project seeks to resolve the long-wavelength limit of oxygenic photosynthesis in order to constrain the range of extrasolar environments in which spectral signatures of biogenic oxygen might be found, and thereby guide future planet detecting and characterizing observatories.

  ROADMAP OBJECTIVES: 5.1 6.1 6.2 7.2

• Neoproterozoic Carbon Cycle

  The rock record late Neoproterozoic (540-800 Ma) appears to exhibit strong
  perturbations to Earth’s carbon cycle. This project seeks an understanding
  of the mechanisms that drive such events and their biogeochemical significance.

  ROADMAP OBJECTIVES: 3.2 4.2 5.1 5.2 6.1

• The Subglacial Biosphere – Insights Into Life-Sustaining Strategies in an Extraterrestrial Analog Environment

  Sub-ice environments are prevalant on Earth today and are likely to have been more prevalent the Earth’s past during episodes of significant glacial advances (e.g., snow-ball Earth). Numerous metabolic strategies have been hypothesized to sustain life in sub-ice environments. Common among these hypotheses is that they are all independent of photosynthesis, and instead rely on chemical energy. Recently, we demonstrated the presence of an active assemblage of methanogens in the subglacial environment of an Alpine glacier (Boyd et al., 2010). The distribution of methanogens is narrowly constrained, due in part to the energetics of the reactions which support this functional class of organism (namely carbon dioxide reduction with hydrogen and acetate fermentation). Methanogens utilize a number of metalloenzymes that have active site clusters comprised of a unique array of metals. During the course of this study, we identified other features that were suggestive of other active and potentially relevant metabolic strategies in the subglacial environment, such as nitrogen cycling. The goals of this project are 1) identifying a suite of biomarkers indicative of biological CH4 production 2). quantifying the flux of CH4 from sub-ice systems and 3). developing an understanding how life thrives at the thermodynamic limits of life. This project represents a unique extension of the ABRC and bridges the research goals of several nodes, namely the JPL-Icy Worlds team and the ASU-Follow the Elements team.

  ROADMAP OBJECTIVES: 2.1 2.2 5.1 5.2 5.3 6.1 6.2 7.1 7.2

• Viral Ecology and Evolution

  This project is aimed at probing the occurrence and evolution of archaeal viruses in the extreme environments in the thermal areas in Yellowstone National Park. Viruses are the most abundant life-like entities on the planet and are likely a major reservoir of genetic diversity for all life on the planet and these studies are aimed at providing insights into the role of viruses in the evolution of early life on Earth.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2

• The Long Wavelength Limit for Oxygenic Photosynthesis

  Photosynthesis is process where plants and bacteria use solar energy to produce sugar and oxygen. It is also the only known process that produces signs of life (biosignatures) on a planetary scale. And, because starlight (or solar energy) is one of the most common sources of energy, it is expected that photosynthesis will be successful on habitable extrasolar planets. Our team is studying how photosynthetic pigments – the molecules that make photosynthesis possible – might function in unique or extreme environments on other planets. In our experiments, we use a bacteria called Acaryochloris marina to study how different photosynthetic pigments work. This bacterium is useful for our research because it uses a pigment known as chlorophyll d instead of chlorophyll a, which is more common on our planet. Chrolophyll a works well in Earth’s environment but, by studying chlorophyll d, we can begin to understand how photosynthesis might work on planets with different environments than Earth. So far, our research is revealing that photosynthesis can occur quite efficiently in environments that are very different from our planet.

  ROADMAP OBJECTIVES: 3.2 4.2 5.1 5.3 6.2 7.2

• Project 4A: Characterization of Novel Solid-Phase Fe(II)-Oxidizing Chemolithotrophic Bacteria From Subsurface Environments

  Ferrous iron (Fe(II)) can serve as an energy source for a wide variety of chemolithotrophic microorganisms (organisms that gain energy from metabolism of inorganic compounds). Fe(II) oxidation may have played a role in past (and possibly, present) life on Mars, whose crust is rich in primary Fe(II)-bearing silicate minerals, as well as Fe-bearing clay minerals formed during weathering of primary silicates. This project examined the physiological and phylogenetic properties of novel solid-phase Fe(II)-oxidizing bacteria (FeOB) isolated from subsurface sediments from the Columbia River basin in eastern Washington, as well as clay-rich subsoils from Madison, WI. The organisms were enriched using biotite (a Fe(II)-bearing primary silicate mineral) as an energy source, and purified on organics-containing medium. The capacity of the isolates to oxidize soluble and solid-phase Fe(II) compounds was assessed, and the 16S rRNA gene sequence for each of the isolate was determined. The results revealed that a wide variety of Proteobacteria are capable of catalyzing solid-phase Fe(II) oxidation, including several groups of organisms not previously known as FeOB. These results confirm and expand our knowledge of solid-phase chemolithotrophic Fe(II) oxidation on Earth, and bolster the concept of mineral-associated Fe(II) oxidation as a potential basis for microbial life on other terrestrial planets.

  ROADMAP OBJECTIVES: 3.2 5.1

• Stoichiometry of Life, Task 2a: Field Studies – Yellowstone National Park

  Field work and subsequent laboratory analysis is an integral part of following the elements. One of our field areas is the hot spring ecosystems of Yellowstone, which are dominated by microbes, and where reactions between water and rock generate diverse chemical compositions. These natural laboratories provide numerous opportunities to test our ideas about how microbes respond to different geochemical supplies of elements. Summer field work and lab work the rest of the year includes characterizing the natural systems, and controlled experiments on the effects of changing nutrient and metal concentrations (done so as to not impact the natural features!).

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2 7.2

• Stoichiometry of Life, Task 2b: Field Studies – Cuatro Cienegas

  Cuatro Cienegas is a unique biological preserve in México (state of Coahuila) in which there is striking microbial diversity, potentially related to extreme scarcity of phosphorus. We aim to understand this relationship via field sampling of biological and chemical characteristics and a series of enclosure and whole-pond fertilization experiments.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2

• Understanding Past Earth Environments

  For much of the history Earth, life on the planet existed in an environment dramatically different than that of modern-day Earth. Thus, the ancient Earth represents a planet with a biosphere that is both dramatically different than the one in which we live and is accessible to detailed study. As such, is serves as a model for what types of biospheres we may find on other planets. A particular focus of our work was on the “Early Earth” (formation through to about 500 million years ago), a timeframe poorly represented in the geological and fossil records but comprises the majority of Earth’s history. We have studied the composition of the ancient atmosphere, modeled the effects of clouds on such a planet, studied the sulfur, oxygen and nitrogen cycles, and the atmospheric formation of molecules that were likely important to the origins of life on Earth.

  ROADMAP OBJECTIVES: 1.1 1.2 4.1 4.2 5.1 5.2 6.1

• Stoichiometry of Life, Task 3b: Ancient Records – Genomic

  The goal of Task 3b is to advance understanding of elemental cycling in ancient ecosystems. Team members are developing experimental and computational approaches aimed at genomic analysis of modern ecosystems, and extending these approaches in novel ways to infer the function and composition of ancient communities.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3

• Project 6A: Astrobiology and Habitability Studies Supporting Mars Research and Missions

  Field research at Mars analogs sites such as desert environments can provide important constraints for instrument calibration and landing site strategies of robotic exploration missions to Mars that will investigate habitability and life beyond Earth during this decade. We report on astrobiology field research from the Mars Desert Research Station (MDRS) in Utah Hanksville conducted during the EuroGeoMars 2009 campaign. EuroGeoMars 2009 was an example of a Moon-Mars field research campaign dedicated to the demonstration of astrobiology instruments and a specific methodology of comprehensive measurements from selected sampling sites. Special emphasis was given to sample collection and pre-screening using in-situ portable instruments. We have investigated 10 selected samples from different geological formations including Mancos Shale, Morrison, and Dakota Formation as well as a variety of locations (surface, subsurface and cliffs) partly in-situ in the habitat or in a post-analysis cycle. We compiled the individual studies and tried to establish correlations among environmental parameters, minerals, organic markers and biota. The results are interpreted in the context of future missions that target the identification of organic molecules and biomarkers on Mars.

  ROADMAP OBJECTIVES: 2.1 5.1

• Project 7C: Improving Accuracy of in Situ Stable Isotope Analysis by SIMS

  Isotopic analysis of biologically important elements in petrographic context is a fundamental tool for astrobiology. Secondary ion mass spectrometry (SIMS) is a powerful tool used to understand biogeochemical processes at the spatial scale of the individual microorganisms that drive them. The benefits of the extremely high spatial resolution offered by SIMS do not come without costs, however. Unconstrained physical and chemical variables in the sample of interest can introduce biases leading to inaccurate measurements. Understanding and constraining these barriers to accuracy as we move towards ever finer spatial resolution and analytical precision is a primary focus at WiscSIMS.

  ROADMAP OBJECTIVES: 4.1 5.1 5.2 5.3 7.1