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Project Reports

• Life Underground

  Our multi-disciplinary team from USC, Caltech, JPL, DRI, RPI, and now also Northwestern is developing and employing field, laboratory, and modeling approaches aimed at detecting and characterizing microbial life in the subsurface—the intraterrestrials. We posit that if life exists, or ever existed, on Mars or other planetary body in our solar system, evidence thereof would most likely be found in the subsurface. This study takes advantage of unique opportunities to explore the subsurface ecosystems on Earth through boreholes, mine shafts, sediment coring, marine vents and seeps, and deeply-sourced springs. Access to the subsurface—both continental and marine—and broad characterization of the rocks, fluids, and microbial inhabitants is central to this study. Our focused research themes require subsurface samples for laboratory and in situ experiments. Specifically, we are carrying out in situ life detection, culturing and isolation of heretofore unknown intraterrestrial archaea and bacteria using numerous novel and traditional techniques, and incorporating new and existing data into regional and global metabolic energy models.

  ROADMAP OBJECTIVES: 2.1 2.2 3.1 3.3 4.1 5.1 5.2 5.3 6.1 6.2 7.2

• Astrobiology of Icy Worlds

  Our goal in the Astrobiology of the Icy Worlds Investigation is to advance our understanding of the role of ice in the broad context of astrobiology through a combined laboratory, numerical, analytical, and field investigations. Icy Worlds team pursues this goal through four major investigations namely, the habitability, survivability, and detectability of life of icy worlds coupled with “Path to Flight” Technology demonstrations. A search for life linked to the search for water should naturally “follow the ice”. Can life emerge and thrive in a cold, lightless world beneath hundreds of kilometers of ice? And if so, do the icy shells hold clues to life in the subsurface? These questions are the primary motivation of our science investigations

  ROADMAP OBJECTIVES: 1.1 2.1 2.2 3.1 3.2 4.1 5.1 5.2 5.3 6.2 7.1

• Project 1A: Field Analog Geology and Astrobiology in Support of Mars Exploration

  We report on preliminary results obtained from the field research campaign exploring the area around the Mars Desert Research Station (MDRS) in Utah (Canyonlands area) in February/March 2012. This region has been previously investigated and characterized as geomorphological and geochemical similar to Mars. Soil and rock samples were collected within two formations of a single geological units, the Brushy Basin and Tununk Shale Member. The objective of this research was to characterize samples from plain, cliff and canyon locations using different analysis techniques including Fourier Transform Infra-Red Spectroscopy (FTIR), X-ray diffraction studies (XRD) and elemental composition surface and morphology (SEM-EDX) in order to determine the sample mineralogy and its variability within the geological unit. The analysis of the organic content of the collected samples (extraction of amino acids) is currently underway. Our aim is to compare data from different formations, topographical units, and specific locations in this Utah desert region in order to determine the variations in chemical and physical properties.

  ROADMAP OBJECTIVES: 2.1 5.3

• Project 1: Dynamics of Self-Programming Systems

  This project is a theoretical attempt to understand how evolution can arise from inanimate physical systems. The key idea is that matter can organize into structures that not only replicate and carry information, but are able to program and reprogram themselves functionally. We have already been able to construct simple computer programs that can increase their complexity in an open-ended way, but in this grant period we have been building a mathematical formulation of how this arises using recursive function theory. We have also been trying to develop cellular automata meta-programming pairs that can co-evolve complexity.

  ROADMAP OBJECTIVES: 3.2 4.1 4.2 5.3 6.2

• Biogenic Gases From Anoxygenic Photosynthesis in Microbial Mats

  This lab and field project aims to measure biogenic gas fluxes in engineered and natural microbial mats composed of anoxygenic phototrophs and anaerobic chemotrophs, such as may have existed on the early Earth prior to the advent of oxygenic photosynthesis. The goal is to characterize the biogeochemical cycling of S, H, and C in an effort to constrain the sources and sinks of gaseous biosignatures that may be relevant to the detection of life in anoxic biospheres on habitable exoplanets.

  ROADMAP OBJECTIVES: 4.1 5.2 5.3 6.1 6.2 7.2

• Mineralogical Traces of Early Habitable Environments

  The goal of our work is to understand how habitability (potential to support life) varies across a range of physical and chemical parameters, in order to support a long-term goal of characterizing habitability of environments on Mars. The project consists of two main components: 1. We are examining the interplay between physicochemical environment and associated microbial communities in a subsurface environment dominated by serpentinization (a reaction involving water and crustal rocks, which indicated by surface mineralogy to have occurred on ancient Mars). 2. We are working to understand how mineral assemblages can serve as a lasting record of prior environmental conditions, and therefore as indicators of prior habitability. This component directly supports the interpretation of mineralogy data obtained by the CheMin instrument on the Mars Science Laboratory.

  ROADMAP OBJECTIVES: 2.1 5.3

• Coupled Energy Balance Ecosystem-Atmosphere Modeling of Thermodynamically-Constrained Biogenic Gas Fluxes Project

  The thermodynamically-constrained fluxes of gases to and from a biosphere has profound, planet-wide consequences. These fluxes can directly control the redox state of the surface environment, the atmospheric composition, and the concentration of nutrients and metals in the oceans. Through these direct effects, they also create strong forcings on the climate, the redox state of the interior of the planet, and the detectability of the biosphere by remote observations. This is a theoretical modeling study to constrain biomass, productivity, and biogenic gas fluxes given a range of geologic parameters.

  ROADMAP OBJECTIVES: 1.1 1.2 5.2 5.3 6.1 7.2

• Project 1D: Iron Biogeochemistry in Chocolate Pots Hot Spring, Yellowstone National Park

  Small cores were collected from six locations along a transect following the main fluid flow path at Chocolate Pots (CP) hot spring, Yellowstone National Park. The cores were sectioned at 1 cm intervals, and the solids subjected to sequential extraction to isolate different Fe pools. The results showed that cores proximal to the vent outlet contained significant quantities of dissolved/colloidal and HCl-extractable reduced (ferrous) iron [Fe(II)]. Fe recovered from the other cores was present entirely as Fe(III). The most likely explanation for these observations is that internal generation of Fe(II) via microbial reduction is taking place in deposits proximal to the vent. This interpretation is consistent with rapid Fe(II) production during anaerobic incubation of near-vent deposits. Our results provide direct evidence of Fe(III) oxide reduction in deposits proximal to the main vent at CP, and to our knowledge represent the first demonstration of in situ Fe(III) reduction in a circumneutral-pH geothermal environment analogous to those which may have been present on the ancient Earth and Mars. Preliminary stable Fe isotope measurements on the dissolved/colloidal and 0.5M HCl-extractable Fe fractions in the CP cores suggests that Fe(III) reduction influences the isotopic composition of Fe phases proximal to the vent. A comprehensive analysis of all Fe phases in the cores is underway and will be used to develop conceptual models of controls on the stable Fe isotope composition preserved in the hot spring deposits.

  ROADMAP OBJECTIVES: 2.1 4.1 5.3 7.1

• Project 4: Rapid Evolution in Stressed Populations: Theory

  Evolution is typically thought of as occurring over millions of years. But recently it has become clear that we have grossly over-estimated this time scale. Perhaps the most famous example of this is the rapid evolution of resistance of bacteria, worldwide, to modern antibiotics. Similarly, early life has an evolution time scale problem: given the age of the Earth and the known age of the Last Universal Common Ancestor, life must have arisen and evolved the majority of the complexity of the modern cell in less than a billion years. This project is a theoretical attempt to understand how a fluctuating environment can accelerate evolution rate, and lead to evolution on ecosystem time scales. Eventually, this work will join up with the experimental work being done by our team, using the GeoBioCell, in Project 8.

  ROADMAP OBJECTIVES: 4.1 4.2 5.1 5.2 5.3 6.1

• Project 1E: Microbial Communities in Chocolate Pots Hot Spring, Yellowstone National Park

  DNA was extracted from samples obtained from cores collected at six locations along a transect following the main fluid flow path at at Chocolate Pots (CP) hot spring, Yellowstone National Park. 454 pyrosequencing of 16S rRNA gene amplicons was performed on the extracts, resulting in the generation more than 70 amplicon libraries, each containing a between ca. 2500 and 7500 ca. 300 base pair-long reads. The raw reads were processed and analyzed for their phylogenetic affiliation and other comparisons using the QIIME pipeline. The results indicate that microbial communities in the upper few cm of the Fe/Si-rich CP deposits varied significantly along the sampling transect. Communities at two sites most proximal to the vent source differed substantially from one another and from communities at downstream sites. Although communities at downstream sites were not identical, they were more similar to one another than to the vent-proximal sites. A wide diversity of prokaryotic taxa, including both Bacteria and Archaea, were identified in the libraries, many of which are only distantly (e.g. <90% similarity in 16S rRNA gene sequence) related to known taxa. Communities in cores close to the vent were dominated by anaerobic taxa, many of which have the potential to function as Fe(III) reducers. This result is consistent with the relatively high abundance of reduced (ferrous) iron [Fe(II)] and the rapid rate of Fe(II) production observed in in vitro Fe(III) reduction experiments with material from sites near the vent. Abundant taxa at downstream sites included organisms related to the known Fe(II)-oxidizing organism Sideroxydans paludicola. These results are consistent with Fe geochemical data, which indicate that Fe(II) oxidation is likely the dominant Fe redox cycling pathway in deposits more than 1-2 meters from the vent source. A detailed metagenomic analysis of communities in the upper 1 cm at three sites is underway, with the goal of confirming the function of recognized taxa, and revealing the identity and function of potentially novel Fe redox cycling taxa.

  ROADMAP OBJECTIVES: 2.1 4.1 5.1 5.3 7.1

• Project 5: The Origins of Life’s Diversity

  The huge diversity of life poses a major challenge to ecological theory and a major source of optimism for astrobiology. Ecological theory argues that a single environmental niche should be colonized by a single species of organism, or perhaps a small community, and so the diversity of life should be essentially a measure of the number of niches present. The huge diversity of life does suggest, however, that the ability of life to explore, colonize and especially create environmental niches has been drastically underestimated. Accordingly, the likelihood of extraterrestrial life arising is also underestimated, or at least inadequately estimated, by our present understanding of biological evolution. This project attempts to solve this problem by developing a new theory for niche diversity.

  ROADMAP OBJECTIVES: 3.4 4.1 4.2 5.1 5.2 5.3 6.1 6.2

• Project 1F: Chemolithotrophic Microbial Communities in Subglacial Sediments

  Recent interpretation of Yellowknife Bay, Gale Crater, Mars as an ancient lake basin characterized by low salinity, circumneutral pH, and Fe and S compounds in a range of redox states (1) motivates inquiry into the capability of analogous Earth systems to support microbiomes founded on Fe and S chemolithoautotrophy. The research progress outlined herein was conducted to improve understanding of the microbial metabolisms that promote Fe and S redox transformation in an analogous system – the subglacial environment Robertson Glacier (RG), Peter Loughleed Provincial Park, Alberta, Canada. We seek to better understand the mechanisms by which chemolithoautotrophs access mineral-bound electron donors and acceptors and the potential for biosignature preservation associated with this type of life. Geochemical attributes of the RG subglacial environment that are consistent with the former aqueous habitat at Yellowknife Bay include circumneutral pH, low salinity, and sulfur (S) and iron (Fe) existing in a range of oxidation states. Further, the structure, composition, and function of the endogenous subglacial microbiome at RG is largely shaped by redox transformation of pyrite (FeS₂) and chemolithoautotrophic growth on released Fe and/or S intermediates. To achieve these goals we have assembled a collaborative, multidisciplinary team with expertise in molecular biology, microbial physiology, geochemistry, and thermodynamics.

  ROADMAP OBJECTIVES: 4.2 5.3 7.2

• Biosignatures of Life in Ancient Stratified Ocean Analogs

  Instigated by Macalady and Kump in 2010, this project investigates biosignatures of life in modern analogs for stratified ancient and/or extraterrestrial oceans. The primary field site is a sinkhole in Florida. Other field site include stratified ocean analogs in the Bahamas, New York State, and the Dominican Republic. A website monitoring the activities of an informal working group on Early Earth Photosynthesis is maintained by Macalady (http://www.geosc.psu.edu/~jlm80/EEP.html).

  ROADMAP OBJECTIVES: 2.1 3.3 3.4 4.1 5.2 5.3 6.1 7.1 7.2

• Project 6. Mining Archaeal Genomes for Signatures of Early Life: Comparison of Metabolic Genes in Methanogens

  Methanogenic archaea derive energy from simple starting materials, producing methane and carbon dioxide in the process. The chemical simplicity of the growth substrates and versatility of the organisms in extreme environments provide for a possibility that they could exist on other planets. By characterizing the evolution of methanogens from the most simple to most complex organism as well as their growth characteristics under controlled environments, we hope to address the question as to whether they could exist on planets such as Mars, where bursts of methane have been seen, yet no source has yet been identified.

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

• Biosignatures of Life in Extremely Energy-Limited Environments

  The terrestrial subsurface is the least explored habitat on earth and is characterized by darkness and reducing conditions that limit how fast microbes can obtain energy (low energy fluxes). The diversity and metabolic strategies of microbes in this environment are the subject of our investigation.

  ROADMAP OBJECTIVES: 4.1 4.3 5.1 5.3 7.1

• Jon Toner NAI NPP Postdoc Report

  Aqueous salt solutions are critical for understanding the potential for liquid water to form on icy worlds and the presence of liquid water in the past. Salty solutions can form potentially habitable environments by depressing the freezing point of water down to temperatures typical of Mars’ surface or the interiors of Europa or Enceladus. We are investigating such low-temperature aqueous environments by experimentally measuring the low temperature properties of salt solutions and developing thermodynamic models to predict salt precipitation sequences during either freezing or evaporation. These models, and the experimental data we are generating, are being applied to understand the conditions under which water can form, the properties of that water, and what crystalline salts indicate about environmental conditions such as pH, temperature, pressure, and salinity.

  ROADMAP OBJECTIVES: 2.1 5.2 5.3

• Titan as a Prebiotic Chemical System – Benner

  In 2007, NASA sponsored a committed of the National Academies of Science to explore whether life might exist in environments outside of the traditional habitable zone, defined as positions in a solar system where liquid surface water might be found. Alternative solvents which have analogous “habitable zones” farther away from their star include hydrocarbons, ammonia, and dinitrogen. The core question asked whether life having genetic biopolymers might exist in these solvents, which are in many cases (including methane) characterized by the need for “cold” (temperatures < 100K in the case of methane).

  These “weird” solvents would require “weird” genetic molecules, “weird” metabolic processes, and “weird” bio-structures. In pursuit of this “big picture” question, we turned to Titan, which has exotic solvents both on its surface (methane-hydrocarbon) and sub-surface (perhaps super-cooled ammonia-rich water). This work sought genetic molecules that might support Darwinian evolution in both environments, including non-ionic polyether molecules in the first and biopolymers linked by exotic oxyanions (such as phosphite, arsenate, arsenite, germanate) in the second.

  In the current year, we completed our studies that identified biopolymers that might work in hydrocarbon solvents. These studies have essentially ruled out biological processes in true cryosolvents. However, a series of hydrocarbons containing different numbers of carbon atoms (one, two, three, and four, for example, in methane, ethane, propane, and butane) cease to be cryosolvents as their chain lengths increase. These might be found on “warm Titans”. Further, they might exist deep in Titan’s hydrocarbon oceans, where heating from below would lead to warm hydrocarbon oceans.

  These studies showed that polyethers are insufficiently soluble in hydrocarbons at very low temperatures, such as the 90-100 K found on Titan’s surface where methane is a liquid at ambient pressures. However, we did show that “warm Titans” could exploit propane (and, of course, higher hydrocarbons) as a biosolvent for certain of these “weird” alternative genetic biopolymers; propane has a huge liquid range (far larger than water). Further, we integrated this work with mineralogy-based work that allows reduced molecules to appear as precursors for less “weird” genetic biomolecules, especially through interaction with various mineral species, including borates, molybdates, and sulfates.

  ROADMAP OBJECTIVES: 1.1 1.2 2.2 3.1 3.2 3.4 4.1 5.3 6.2 7.1 7.2

• Project 9: Evolution Through the Lens of Codon Usage

  The sequences of protein encoding genes are subject to multiple levels of selection. First, amino acid changes that adversely alter protein function are unlikely to survive. In addition, the genetic code of organisms is degenerate; it includes alternative (synonymous) codons for most of the amino acids. Codon usages in a genome are generally viewed as a balance between drift and selection for rapid and accurate translation of mRNAs into proteins. This balance defines the native codon usage of the organism. Later, it was recognized that many horizontally transferred genes have distinctive codon usages. It was assumed that these reflected the codon usages of the organisms that contributed the genes. We viewed this as an opportunity to identify those sources.

  These studies of this have led us to discover that: (i) most of the recently acquired genes come from such closely related organisms that their distinctive codon usages cannot be attributed to a phylogenetically distant source; (ii) the transfers commonly exceed recognized boundaries of microbial species; (iii) after their acquisition, some of the genes do not drift to match the native codon usage of the recipient; (iv) many of the genes that are most up-regulated under starvation conditions have this same codon usage; and (v) a distinctive stress/starvation-associated codon usage is a recurring theme that is observed in diverse Bacteria and Archaea.

  These studies entailed the development of a variety of new codon usage analysis tools. We are making these tools available, and are integrating them into the RAST genome annotation and analysis server at Argonne National Laboratory.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3

• Stoichiometry of Life – Task 1 – Laboratory Studies in Biological Stoichiometry

  This project component involves a set of studies of microorganisms with which we are trying to better understand how living things use chemical elements (nitrogen, phosphorus, iron, etc.) and how they cope, in a physiological sense, with shortages of such elements. For example, how does the “elemental recipe of life” change when an organism is starved for phosphorus or nitrogen or iron? Is this change similar for different species of microorganisms? Are the changes the same if the organism is limited by a different key nutrient? Furthermore, how does an organism shift its patterns of gene expression when it is starved by various nutrients? This will help in interpreting studies of gene expression in natural environments.

  ROADMAP OBJECTIVES: 5.2 5.3 6.1 6.2

• The Long Wavelength Limit of Oxygenic Photosynthesis

  Oxygenic photosynthesis (OP) produces the strongest biosignatures at the planetary scale on Earth: atmospheric oxygen and the spectral reflectance of vegetation. Both are controlled by the properties of chlorophyll a (Chl a), its ability to perform the water-splitting to produce oxygen, and its spectral absorbance that is limited to red and shorter wavelength photons. We seek to answer what is the long wavelength limit at which OP might remain viable, and how. This would clarify whether and how to look for OP adapted to the light from stars redder than our Sun.

  Previously under this project, with other co-investigators we spectrally quantified the thermodynamic efficiency of photon energy use in the chlorophyll d utilizing cyanobacterium, Acaryochloris marina str. MBIC11017, determining that it is more efficient than a Chl a cyanobacterium. The current focus of the project is aimed at understanding the adaptations of far-red/near-infrared (NIR) oxygenic photosynthetic organisms in general: what is their ecological niche where they are competitive against chlorophyll a organisms in nature, and what energetic shifts have been made in their photosynthetic reactions centers to enable their use of far-red/NIR photons. Field sampling and measurements are being conducted to isolate new strains of far-red utilizing oxygenic photosynthetic organisms, to quantify the spectral and temporal light regime in which they and previously discovered strains live in nature, and use these light measurements to drive kinetic models of photon energy use to ascertain light thresholds of survival.

  ROADMAP OBJECTIVES: 3.2 4.2 5.1 5.3 6.2 7.2

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

  Yellowstone National Park harbors an array of hydrothermal ecosystems with widely varying geochemical characteristics and microbial communities. Our research aimed to understand how the geochemistry of these hot springs shapes their constituent microbial communities including their composition and function. To accomplish this aim, we measured (1) physical and geochemical properties of hot spring fluids and sediments, (2) the rates of biogeochemical processes (i.e., methane oxidation, nitrogen fixation, microbial Fe cycling, photosynthesis, de-nitrification, etc.), and (3) markers for microbial community diversity (i.e., SSU rRNA, metabolic genes, lipids, proteins).

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2 7.2

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

  We performed two studies to evaluate ecological impacts of nitrogen and/or phosphorus fertilization in a P-deficient and hyperdiverse shallow pond in the valley of Cuatro Cienegas, Mexico.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2

• Project 2G: Iron Isotope Fractionations Among Oxide Minerals Under Acidic Conditions

  The study of Fe isotope exchange and fractionation between aqueous Fe(II) and goethite was motivated by the inferred acidic environment for early Mars, where iron oxides (i.e. jarosite, goethite) were likely present. We found that the extent of atom exchange positively correlates with increasing pH during interactions between Fe(II)_aq and goethite. The decrease in extent of exchange correlates with a decrease in the amount of sorbed Fe(II) to the goethite surface, which strongly suggests that sorbed Fe(II) is the primary catalyst for inducing Fe isotope exchange. The slow rate of isotopic exchange at acidic pH suggests that stable Fe isotope compositions may be resistant to change in acidic aqueous environments, thus leading to preservation of signatures that might contribute to the understanding of ancient Mars paleoenvironments.

  ROADMAP OBJECTIVES: 2.1 5.3 7.1 7.2

• Stoichiometry of Life – Task 2c – Field Studies – Other

  We performed biogeochemical and microbiological studies of novel aquatic habitats, floating pumice in lakes of northern Patagonia that were derived from the 2011 eruption of the Puyehue / Cordon Caulle volcano in Chile.

  ROADMAP OBJECTIVES: 4.1 5.2 5.3 6.1

• Project 13: Experimental Determination of the Existence of the Darwinian Transition

  Life on our planet can be divided into three domains: Archaea, Bacteria and Eukarya. While some genes may be shared among the domains of life, others especially those involved in information processing namely DNA replication, transcription and translation are often unique to a particular domain. It has, therefore, been proposed that the molecular machineries that carry out these processes (replication, transcription and translation) have crossed a so-called Darwinian threshold where the molecular machineries have become gelled and therefore intolerant of new components. This project is examining the Darwinian threshold hypothesis by testing the interchangeability of the components of the DNA replication machinery across the domains of life. Further experiments will examine the capacity of biomolecules involved in translation and transcription to substitute for their counterparts across the domains of life.

  ROADMAP OBJECTIVES: 3.2 3.4 4.2 5.3

• Task 3b: Ancient Records – Genomic

  Task 3b team members are involved in deciphering genomic records of modern organisms as a way to understand how life on Earth evolved. At its core, this couples the integrated measurement and modeling of evolutionary mechanisms that drove the differences between extant genomes (and metagenomes), with experimental data on how environmental dynamics might have shaped these differences across geological timescales. This goal draws from team members’ expertise encompassing theoretical and computational biology, microbial evolution, and studying life in both extreme and dynamic environments across the planet.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3