The Astrophysics of life: Exoplanets and extremophiles _______________________________ Principal Investigator I. N. Reid Space Telescope Science Institute* 3700 San Martin Drive Baltimore, MD 21218 *operated by Association of Universities for Research in Astronomy, Inc. (AURA) _________________________________ Co-Investigators Space Telescope Science Institute C. Christian D.R. Soderblom M. Livio W.B. Sparks A. Nota J. Valenti M. Postman Princeton University E.L. Turner C.F. Chyba N.J. Kasdin M. Littman Center of Marine Biotechnology, University of Maryland S. DasSarma F. Robb F. Chen K. Sowers Carnegie Institution of Washington M. Turnbull B. Paczynski D. Spergel S. Tremaine R. Vanderbei _______________________________ Collaborators and Consultants Space Telescope Science Institute R. Gilliland P. McCullough S. Lubow K. Sahu University of Maryland J. Coker A. Frederick A. Colman Carnegie Institution of Washington M. Fogel A. Steele Princeton University T.C. Onstott University of New South Wales R. Cavicchioli Morgan State University J. Müller NIST T. Germer Idaho State University L. DeVeaux National Astronomical Observatory of Japan M. Tamura T. Yamada Smithsonian Astrophysical Observatory J. Winn University of Melbourne S. Wyithe Oxford Brookes University S. McCready Tokyo University Y Suto Jet Propulsion Laboratory R. Carlson Notice of Restriction on Use and Disclosure of Proposal Information The information (data) contained in Volume 2, Section 6 (Budget) of this proposal constitutes a trade secret and/or information that is commercial or financial and confidential or privileged. It is furnished to the Government in confidence with the understanding that it will not, without permission of the offeror, be used or disclose other than for evaluation purposes; provided, however, that in the event a contract (or other agreement) is awarded on the basis of this proposal, the Government shall have the right to use and disclose this information to the extent provided in the contract (or other agreement). This restriction does not limit the Government’s right to use or disclose this information (data) if obtained from another source without restriction. Confidential Page 0 2/12/2016 Executive Summary ........................................................................................................................ 2 Summary of Personnel, Commitments and Cost ............................................................................ 5 1. Research and Management Plan: Introduction ........................................................................... 6 2. Where are the exoplanets? .......................................................................................................... 9 3. What are the intrinsic and extrinsic environments of exoplanets? ........................................... 22 4. Can the exoplanets support life? ............................................................................................... 29 5. How will we recognize if life is present?.................................................................................. 47 6. Management Plan...................................................................................................................... 61 7. Strengthening the NAI Community .......................................................................................... 66 8. Education, Outreach and Press Presence .................................................................................. 73 9. References ................................................................................................................................. 83 10. Appendix: Glossary of terms and acronyms ........................................................................... 97 Confidential Page 1 2/12/2016 Executive Summary Synopsis: A team of leading scientists and engineers located at the Space Telescope Science Institute (STScI, lead institution), the Center of Marine Biotechnology (COMB) at the University of Maryland Biotechnology Institute and in the School of Engineering and Department of Astrophysical Sciences at Princeton University (PU) will collaborate to form a new NAI node focused on the scientific goal of identifying habitable exoplanets, the collaborative purpose of expanding the NAI’s connections to and between the exoplanet and extremophile research communities and a suite of outreach/education/training projects targeted on diverse audiences, including the general public, K-12 level students and teachers, PU undergraduates and the members of relevant scientific communities. The ultimate purpose of the team’s efforts is to expand our understanding of the potential for life in environments beyond, and probably even more extreme than, those present on Earth and in the Solar System. Unifying Intellectual Themes and Focus: Research on extremophiles, particularly psychrophiles, and on exoplanetary systems are the two major scientific themes of the proposed research program and are supplemented by some investigations of Solar System objects that seem particularly likely to provide useful lessons concerning the astrobiological significance of extremophiles and for future astrobiological investigations of exoplanets. Although a diverse set of astronomical and biological research projects are proposed, all are connected to the goal of identifying habitable planets via their relevance to one of the four major questions around which the overall effort is organized: 1 - Where are the exoplanets? 2 - What are their intrinsic and extrinsic environments? 3 - Can they support life? 4 - How will we recognize if life is present? These four questions are addressed by projects described in sections 2, 3, 4 and 5 (respectively) of the Research and Management Plan and each is the topic of major research efforts centered in either two or all three of the participating institutions, and through a collaboration with scientists at other NAI nodes and beyond. Thus the proposed research, in turn, directly addresses three Astrobiology Roadmap goals: #1 – “Understand the nature and distribution of habitable environments in the Universe”, #5 – “Understand the evolutionary mechanisms and environmental limits of life”, and #7 – “Determine how to recognize signatures of life on other worlds and on early Earth”. It also indirectly addresses important aspects of a fourth goal: #2 – “Explore for past or present habitable environments, pre-biotic chemistry and signs of life elsewhere in the Solar System”. These issues are central to “Earth-like Planets and Life”, one of the four key research areas in NASA’s Vision for Space Exploration. Research Program: The research programs associated with each of the four questions listed above are summarized below: 1. Where are the exoplanets? We will compile a Solar Neighborhood Habitability Census (SNHC), and use this investigative tool to determine the potential for habitable worlds around other stars. This compendium will go beyond the conventional paradigm, paying particular attention to stars with lower mass than the Sun, notably M dwarfs (contributing to the SETI Institute NAI Team’s M-star initiative), and binary systems. We will provide comprehensive, Confidential Page 2 2/12/2016 carefully vetted information on both the stars themselves and their immediate environments. The compilation will focus on the nearest stars, but will include all known exoplanetary systems, including new results from ground and space based observations (e.g., microlensing surveys, high precision spectroscopy of transiting “hot Jupiter” systems, Kepler GO observing programs). We will use the SNHC to clarify what is known about the Sun’s neighbors as potential hosts of habitable exoplanets. We will identify stars that lack critical observations and, where possible, acquire the appropriate data through supplementary observations. In particular, the PU coronagraphic optics group is developing high-contrast imaging techniques that will be invaluable in identifying faint companions of nearby stars from the ground and in studying terrestrial exoplanets directly via future space missions. We will combine the observed stellar properties with constraints on life-limiting conditions, including those derived from experiments on extremophiles proposed here, to estimate the likelihood that individual exoplanet systems are capable of harboring habitable exoplanets. 2. What are their intrinsic and extrinsic environments? We know that planetary environments depend on the properties and behavior of the central star, the planet’s orbital characteristics and the exoplanet atmospheric and surface properties. Terrestrial exoplanets are likely to remain below the detection threshold for direct imaging for at least a decade, although they should be detected in both radial velocity and transit surveys (the Kepler mission); gas giants, however, are potentially accessible to direct imaging, while their atmospheric properties are already being measured for transiting systems. Such observations are invaluable in determining whether exoplanets, including “hot Jupiters” (gas giants on ~3-day orbits around solar-type stars), closely resemble gas giants in our Solar System. We will pursue further observations of planetary exospheres, capitalising on results from transit surveys and high-contrast coronagraphy. Specific observing programs will utilize the Hubble Space Telescope, very high resolution and signal-tonoise spectroscopy with the Subaru 8.2-meter telescope and large telescope spectroscopic and photometric surveys. We will also investigate the dynamical evolution of exoplanetary systems, particularly examining the distribution of orbital eccentricity and long-term dynamical stability. 3. Can they support life? Not all exoplanets, and perhaps very few, will be able to support life. We will match our assessment of the likely conditions on exoplanets against terrestrial life’s experimentally determined limits; this requires a reliable set of metrics for the limiting environments which allow life’s survival and growth. Determining the low temperature limit for life is particularly important, since cold environments are likely to be far more common than warm ones. This holds even for Earth, where 80% of the biosphere by volume exists at average temperatures below 5o C. We will build on a heritage of over 2 years of experiments by the COMB and STScI teams designed to examine cold limits for life, and investigate how life adapts physiologically (e.g. through biofilm formation) and genetically (e.g., by induction of molecular chaperones) to various types of extreme environments (see Reid et al, 2006). We will also investigate the effects of cyclic variations in conditions, since many known exoplanets are on elliptical orbits. Finally, we will examine the radiation sensitivity of extremophile species, and consider the implications for survival in the presence of strong stellar activity, both on planetary surfaces and in space (panspermia). The results from these experiments, along with other studies of extremophiles within the NAI and elsewhere, will provide direct feedback to the SNHC. These results will also be relevant to evaluating the prospects for life on, and planetary protection measures appropriate for, Mars and Europa. Confidential Page 3 2/12/2016 4. How will we recognize if life is present? We will develop innovative methods for the biocharacterization of exoplanets, observable only as faint point sources, a problem crucial to the astrobiological significance of the very rapidly developing astronomical study of exoplanetary systems. Specifically, we propose to conduct a series of observational experiments that will use earthshine to characterize Earth as an effectively unresolved planet. This work will complement and extend studies being carried out by existing NAI nodes (principally those centered at JPL and the University of Arizona). In addition, we will investigate new methods of remote life sensing, notably those based on polarization and high resolution spectroscopic biosignatures. We will test these techniques through observations of Mars, Europa and Enceladus. Education/Public Outreach and Strengthening Astrobiology: In these areas, the team’s efforts will build on existing, highly-successful activities at each of the three participating institutions: STScI has a well established and highly acclaimed suite of programs for providing access to basic information and recent results in astrophysics to both the media and K-12 educators; we will add a major astrobiological component to these activities and use their well established visibility to promote public and student appreciation of the field. PU will expand its current course offerrings in astrobiology and seek to establish a “certificate program” in astrobiology which Princeton undergraduates will be able to attach to their diplomas by satisfying an appropriate set of academic requirements, and PU CoIs will work closely with the existing undergraduate-initiated “astrobiology club” to promote campus interest in the field. A group of Princeton faculty, including all PU CoIs on this proposal, will work to establish an interdisciplinary Center for Planets and Life internal research unit. COMB will evolve its existing annual Extremophile Summer School for graduate students, postdoctoral fellows and other researchers into a five day “Extremophiles in Astrobiology: Theory and Techniques” semiannual summer offerring. This program, in which CoIs from all three institutions will participate, is intended to be symmetrical with (and thus complementary to) the existing NAIsponsored summer programs at Arizona and Hawaii which are intended to familiarize lifescience and Earth-science students with planetary-science and astrophysics. Management Plan: The management structure for the proposed NAI node will be relatively simple but with the necessary formality required to assure productivity given the large, diverse and dispersed nature of the project. In addition to the PI, with overall responsibility, there will be institutional-PIs at COMB and PU who will coordinate local activities and be the primary point of management and administrative contact with the PI and each other. Weekly CoI meetings at each institution and bi-weekly full team video conferencing will provide good communication and encourage close interaction of the various research projects as they progress. Appropriate elements of the overall project (e.g., SNHC, coronograph development, psychrophile characterization) will adhere to a schedule of milestones aimed at producing specific deliverables. A 0.3 FTE Program Manager at STScI, with extensive experience in organizing and steering large and complex technical projects, will assist the PI in matters of budget, schedule and reporting, both within the team and for the team in the larger NAI environment. Finally, the team will be advised by a small, external committee which will convene annually to provide an independent review of all of the team’s activities and initiatives. Confidential Page 4 2/12/2016 Summary of Personnel, Commitments and Cost Commitment (total FTEs) Name Space Telescope Science Institute I. N. Reid Project manager C. Christian M. Livio A. Nota M. Postman D. Soderblom W. Sparks J. Valenti M. Turnbull Postdoctoral fellow Student intern Sr. database engineer Systems administrator Web developer (E/PO) Project manager Animator (E/PO) Informal science educator (E/PO) E/PO evaluator 2.50 1.50 0.50 0.50 0.50 0.50 0.50 0.50 0.25 5.00 5.00 1.92 0.18 0.10 0.48 0.50 0.12 0.32 0.21 Center of Marine Biotechnology S. DasSarma J. Coker F. Chen Graduate student F. Robb A. Colman K. Sowers Graduate student 1.25 5.00 0.50 5.00 0.50 5.00 0.50 5.00 Princeton University E. Turner C. Chyba K. Hand J. Kasdin M. Littman B. Paczynski D. Spergel S. Tremaine R. Vanderbei Graduate students (2.3) Astrophysics/TBN Graduate student Engineering/TBN M. Carr (Sr. Technical Staff) Confidential Page 5 1.25 0.50 1.00 0.50 0.50 0.50 0.50 0.50 0.50 11.5 5.00 1.65 2/12/2016 1. Research and Management Plan: Introduction The central theme of our proposal is identifying habitable exoplanets. This is a central issue for astrobiology, which seeks to address fundamental questions concerning our place in the Universe (see Chyba and Hand, 2005). In this proposal, we take the conventional definition of habitability, and the conventional habitable zone (Kasting, Whitmire & Reynolds, 1993) as a starting point for our investigations, but we explore means of expanding those limits.We also focus primarily on simple microbial species, primarily archaea, and frame our discussions of habitable (and inhabited) worlds largely in that context. The individual research components of this proposal tackle four key questions whose answers work in concert to address this larger purpose: 1) Where are the exoplanets? 2) What are their intrinsic and extrinsic environments? 3) Can they support life? 4) How can we tell if they do? We propose a suite of research projects tied to these four questions. We address the integration of the individual science elements in Section 1.2; here we note that an overarching goal of this proposal, as with the NAI as a whole, is to bring together these different sciences (and scientists) to form a common vocabulary. 1.1 Our team Astrobiology is by its nature interdisciplinary, and a correspondingly interdisciplinary team of scientists has come together to tackle our research program. We include experts on extremophile biology from the Center of Marine Biotechnology (COMB); builders of coronagraphic optics and imaging analysis from Princeton University and Space Telescope Science Institute (STScI); specialists on planetary “demographics,” the constituents of the Solar Neighborhood, and database construction and maintenance from STScI, the Carnegie Institution of Washington (CIW) and Princeton; researchers of planetary dynamics from Princeton and STScI; and experts on Earthshine and planetary biosignatures from Princeton, STScI and CIW. We will be collaborating with researchers from ten other institutions, including Idaho State University, Morgan State University, the University of Tokyo, the University of Melbourne, NIST, JPL, SAO, the National Astronomical Observatory of Japan, the University of New South Wales and Oxford Brookes University. Our team also includes experienced individuals in education, outreach and the public understanding of science, and our E/PO program will be co-ordinated from STScI. In addition, Princeton University is developing a Center for Planets and Life, and offers astrobiology courses at both the undergraduate and graduate level. Graduate students, and, where appropriate, undergraduates from Princeton Unversity, the University of Maryland, Morgan State University, and Johns Hopkins University will be directly involved in the research outlined in this proposal. 1.2 A project synthesis We have proposed a diverse set of projects covering a broad range of biological and astronomical topics. How do these individual projects interact with each other? Figure 1.1 illustrates the cross-fertilization: the first version of the Solar Neighborhood Census (Section 2.2, 2.3) will be created from basic stellar data; initial habitability criteria will be based on the characteristics of the known exoplanets, suggesting targets for further observation; as more exoplanets are identified (Section 2.4), we will refine the SNH Census and the dossier of Confidential Page 6 2/12/2016 exoplanet characteristics (Section 3); the results from our biological experiments on how life adapts to cold and is affected by radiation (Section 4) will likely improve our understanding of the environments of exoplanets and Solar System bodies, and modify our habitability criteria (and the SNH Census, and hence target selection for ground-based observations and space missions); at the same time, analyses and observations of Solar System bodies (Sections 4.4 and 5.5) are likely to influence our biological experiments, and will certainly inform our search for biosignatures (Section 5.2, 5.3 and 5.4) that have potential for future space missions like TPF, Darwin and Lifefinder. Exoplanet detection Solar Neighborhood Habitability Census Characteristics of exoplanets Stellar data Mars, Europa and Enceladus Cold and Life Radiation and Life New biosignatures Figure 1.1 The main modules of our proposed research program: individual modules are color-coded by the underlying question: green – question 1 (Section 2); blue – question 2 (Section 3); yellow – question 3 (Section 4); red – question 4 (Section 5); our proposed research on Mars, Europa and Enceladus is relevant to both questions 3 and 4. We will foster interactions between team members through regular meetings of our full team of CoIs and collaborators. As described in Section 7.6, we plan to offer Extremophile Summer Schools at COMB in years 1, 3 and 5; we will take advantage of those opportunities to hold 1-2 day symposia, where we will present and discuss results from the individual projects. During years 2 and 4, we will hold 3 to 4-day offsite retreats, including representatives from the wider NAI community. 1.3 Research products We propose to produce the following in this program: Confidential Page 7 2/12/2016 First, a major product of our research will be the Solar Neighborhood Habitability Census, an active database that will collect and synthesize key data for stars and their immediate environments. We will use this Census ourselves as an investigative tool for our astrobiological research, but this compilation will also prove invaluable in focusing both ground-based observing campaigns and future NASA missions, including Kepler, the James Webb Space Telescope (JWST), SIM Planetquest, the Terrestrial Planet Finder (TPF) and Lifefinder. A key property of the Census is that it will go beyond merely compiling all available astronomical data for the objects of concern; instead, we will use the acquired knowledge, expertise, and judgment of the team to critically assess and evaluate the available observations, both astronomical and biological, and create a research tool of broad use to the entire astrobiology community. Finally, the Census will be capable of drawing upon the entire research community through adaptability and the inclusion of feedback from others, as well as continuous growth in what the Census includes and how it makes that information available. A second result from this program will be the development and implementation of novel, innovative methods of high-contrast coronagraphic imaging. We will use the associated instrumentation to survey nearby stars for close, faint companions. Third, we will provide new information on how life adapts to low temperature, high radiation and other extreme environments. This work will have clear implications on the prospects for life on Mars and Europa. It will also directly bear on preventing the potential forward contamination of these bodies by microbes that could be present on spacecraft from Earth. Fourth, we will use Solar System objects as reference cases for characterization tools that will be applied to exoplanets. Finally, we will examine Earth as a planet, and identify and characterize new biosignatures suitable for remote life detection. In particular, we will examine the potential of circular polarization as an unambiguous biosignature, sensitive to life at the broadest level, from simple microbial species to complex terrestrial scenes. In addition to these research goals, we are committed to educational and public outreach. Graduate and undergraduate students will be directly involved in our research, thus helping to train the coming generation of astrobiologists. We will share our research experience and results with students and the public at large through innovative educational and informational resources. Our team members bring skills to the NAI that complement the current range of scientific disciplines: in particular, we have Wide-ranging expertise on archaeal extremophiles, particularly cold-adapted species and the biogeochremistry of extreme environments; A profound understanding of the properties of stars in the Solar Neighborhood; In-depth knowledge of cutting-edge high-contrast imaging technology and techniques. Each of these research areas is crucial to successfully identifying habitable, Earth-like planets, and achieving one of the prime goals of NASA’s Vision for exploration. Confidential Page 8 2/12/2016 2. Where are the exoplanets? Goal 1 of the 2002 Astrobiology Roadmap is straightforward: Determine the potential for habitable planets beyond the Solar System, and characterize those that are observable. It is conceivable, of course, that life could exist in the interstellar medium or other, more exotic, locales, but given what we know so far, this Goal boils down to understanding the basic properties of the stars of our Galaxy, how those properties translate into potential habitable zones in the immediate environments of those stars, and how stable those environments remain over the long time scales we associate with biological evolution. We propose to meet this goal through a thorough and comprehensive effort to characterize nearby stars and to derive the astrobiologically important parameters of those stars. To do this we will construct the Solar Neighborhood Habitability Census, a comprehensive database encapsulating key information on the properties of a complete, well-defined stellar sample. As the name suggests, this database focuses on stars near the Sun, but we will also incorporate data for all stars known to have planetary-mass companions. We will not simply amass all available measurements for a given star; rather, we will use the expertise of our team to assess the quality of individual observations and derived parameters, and determine the most reliable measurements for each star in the database. Our main purpose in constructing the SNH Census is to provide an investigative software tool for our own astrobiological research, and to build a bridge from an astrophysical compilation to a facility that our biologist colleagues can draw on to inform their work. The Census will also reach beyond the team of this proposal to the entire astrobiology and NAI community. As we construct the database, we will identify stars that lack crucial data, and, wherever possible, we will obtain the new observations needed to supply the missing information (see Section 2.3.2). Direct detections of planetary companions are of obvious importance, and our team includes key players in several major exoplanet surveys that use a variety of methods, including radial velocities, direct imaging, and photometric monitoring for either transits or microlensing events. In particular, the Princeton Coronagraphic Optics group is developing new high-contrast imaging techniques as a major part of this proposal, and those techniques will be applied to search for low luminosity companions (potentially even gas giant exoplanets) around nearby stars. We will incorporate results from those programs and other planet detection surveys, including those associated with the UCLA and CIW NAI nodes, as they become available. Once in place, the SNH Census will allow us to investigate habitability in a number of ways. For example, Systematic properties of the exoplanet host stars can provide clues to the required conditions for planet formation. The most striking example is the correlation between exoplanet frequency and metallicity (Gonzalez, 1997; Fischer & Valenti, 2005). We can investigate whether the known host stars might harbor habitable exoplanets. Dynamical considerations may rule out the presence of exoplanets with stable orbits in the (conventional) habitable zone (see Section 3.3), while observations of planetary exospheres can provide important information on chemical composition (Section 3.2). Confidential Page 9 2/12/2016 Conversely, we can eliminate field stars that are ill-suited to be habitable exoplanet hosts because of their intrinsic characteristics such as variability, close stellar or brown dwarf companions, or excessive emission of high energy radiation. Applying habitability criteria requires a working definition of life’s limits for survival and growth, an issue tackled by several current NAI programs; some aspects are adressed in Section 4 of this proposal. 2.1 Why a new census? Why undertake this SNH Census at all? Aren’t there already databases for the nearby stars? And what is gained by compiling existing information over new research? Is there value added? Extensive work already exists in nearby-star studies, and a number of compilations of those data are either underway or have been completed recently. These include the NStars database (http://nstars.arc.nasa.gov/index.cfm), the Catalog of Habitable Stellar Systems (HabCat: Turnbull & Tarter, 2003a, b), the TPF target list (http://sco.stsci.edu/tpf_tibdb) and the StARS database. However, none of these is well suited to the needs of the research in this proposal or to the needs of astrobiology in general. First, there is a mismatch in scope. For instance, the NStars database aimed to include all stars within 25 parsecs of the Sun, but the database is no longer maintained, and partial data are available for only a limited subset of stars. The current planet search programs include stars to 40-50 pc, so at best NStars would provide information on a small subset of the stars that have known planetary companions. By comparison, the HabCat database (co-authored by CoI Turnbull) is derived primarily from the Hipparcos and Tycho-2 catalogues, and therefore is dominated by more luminous stars (especially G dwarfs, as explained below), and includes few of the many nearby M dwarfs. The TPF target list focuses specifically on the needs of that NASA mission, and consists mainly of solar-type stars within only 15 parsecs of the Sun. Finally, the full scope of the StARS database, which is still under construction, remains unclear; however, this database is designed to serve a much wider community and a broader range of purposes, while our research is focused specifically on habitability issues. Second, most existing databases amass all the available data for their objects. Our approach is that not all data are created equal, and that a carefully vetted and reviewed census is of much greater value than an uncritical assemblage of everything that may be in print. Our team includes astronomers with career-long familiarity with the different kinds of stars we will include and the observations available for those stars. By applying the knowledge, expertise and judgment of our team we believe we can build a database of higher consistent quality than otherwise possible. Moreover, we do not see ourselves as necessarily the last word, and the SNH Census will provide a means for anyone in the astrobiology community to provide their own assessments. Third, these databases tend to stop at the point that astronomers feel comfortable, which is dealing strictly with astronomical data. A Census intended to serve the astrobiology community should go beyond that to interpret the astrophysical data in terms that are useful to exobiologists, and to extend the database in ways that help make it a tool for the biologists to inform their own work. This is a critical point at which our collaboration between astronomers and biologists really matters and lies at the heart of the concept of an NAI. Confidential Page 10 2/12/2016 Fourth, current databases tend to be conceived in terms of a particular point in time, even if the data itself get updated. In contrast, we are still in the process of identifying subsolar mass M dwarfs within 20 parsecs, and we expect to add more stars to the SNH Census over the duration of this NAI project. We also will update and even expand the database as new or revised relevant measurements become available. This goal is achieved most efficiently with an in-project database. Finally, STScI has well-known strengths in the construction and execution of databases and data archives, notably the observations obtained by HST, but also other missions and other kinds of data products. STScI has the capability to do the SNH Census well. Our overall purpose is to create the SNHC as an investigative tool for our research, not as yet another reference dataset for the general community. Consequently, the database must be responsive to our needs. 2.2 Sampling the Solar Neighborhood The first step in building the SNH Census is the definition of the sample to be included, and the means to identify the members of those families. We will focus on the stars nearest the Sun as our primary sample, with other stars of obvious interest (those with known exoplanets) added for their context. Do these nearest stars adequately represent the Galaxy as a whole? 2.2.1 The Solar neighborhood as a Galactic microcosm Stars near the Sun have long been favorite targets for astronomical research for two main reasons. First, the nearest stars include the brightest members (in apparent magnitude) of any spectral class. These nearby stars provide us with the highest possible photon fluxes for our telescopes, and can therefore be studied in the greatest detail. Also, linear spatial resolution decreases in proportion to the distance of the observed object, so nearby stars offer the best prospects for identifying close (and faint) orbital companions – a key issue for exoplanet surveys. We look at the nearby stars simply because we have the chance to see more. Concentrating on the nearest stars is also important if we desire a well-defined sample with statistically reliable properties. Stellar catalogs become increasingly incomplete with increasing distance, and the effective distance limit for completeness is less for intrinsically fainter stars. Those fainter stars are simply harder to enumerate, of course, among the vast numbers of distant objects in our Galaxy. Distance measurements themselves become less accurate with increasing distance. It is important to bear these limitations in mind when analyzing the average properties (and the distribution of properties) of stellar systems because incomplete samples almost always incorporate hidden or poorly understood biases that can skew the results. Demanding a complete sample restricts our survey to relatively tiny volume of the Milky Way. For example, for sun-like stars, catalogs become incomplete at distances of ~40 parsecs (less than 0.2% the Galactic radius). Despite this, the solar neighborhood in fact includes stars that were born in many different parts of our Galaxy and over the Galaxy’s full history. This is because stars interact gravitationally with each other and with more massive objects, notably Giant Molecular Clouds. Those interactions tend to scatter stellar velocities, so that stars that may have formed together end up well dispersed and well mixed over astronomical time scales. Confidential Page 11 2/12/2016 Not every kind of star is represented in the Solar neighborhood. For example, halo stars (low metallicity members of the Galaxy’s first major stellar population) are extremely rare simply because their number density is low. Similarly, there are few extremely young stars (less than 10 Myrs in age) locally, since the star lies relatively far from active star forming regions. On the other hand, the nearest examples of these systems are under active study by other members of the NAI, including UCLA astronomers (e.g., Zuckerman & Song, 2004) and members of the University of Arizona NAI team. Setting aside these minor exceptions, the local stars are representative of the broad bulk of stars in the Galactic disk, and are therefore likely to provide a fair estimate of the prevalence of habitable environments within the Galaxy as a whole. 2.2.2 Which stars to include? In putting together a representative cross-section of the Galaxy’s stars, it is not sufficient to simply specify a distance limit. Our ideal sample would be complete and volume-limited; i.e., it would include all stars within some specified horizon. In practice, this ideal is neither practical nor feasible. When stars form, the distribution of their masses follows a power law, so that there are huge numbers at the lowest masses, and many fewer at high masses. At the same time, massive stars are inherently much more luminous that stars of lower mass and so can be seen at great distances. Low-mass stars get fainter much faster than they get numerous, so that the M dwarfs comprise fully 80% of our Galaxy by number, yet constitute a mere handful of the 100,000 brightest stars. What this means is that a well-constructed census of the nearby stars will balance these factors to come up with definitions that are best suited to different kinds of stars. This is summarized in Table 2.1: Spectral types1 B, A, F and G K M to L Absolute magnitude MV < +6 +6 < MV < +8 MV > +8 Distance (parsecs) 40 25 20 limit Number systems 2,000 350 2,000 of These choices for distance limits are driven by practical considerations. The first family, of B, A, F, and G stars, is now straightforward to determine because of the precise astrometric observations carried out by the satellite Hipparcos of the European Space Agency. Hipparcos observed essentially all stars brighter than 9th magnitude in V, roughly 75,000 stars. Most of those stars are intrinsically luminous, and too far away for our purposes. Of the remainder, a significant portion is made up of G dwarfs, i.e., stars that are very similar to the Sun. This is because stars more massive than the Sun are inherently less numerous, and, also to some extent because such stars evolve more quickly than the Sun does, so that few are left on the main sequence. Thus the observations made by Hipparcos make it straightforward to list the G dwarfs (and hotter, earlier types) that are within 40 pc. This has already been done by CoI Soderblom as part of his effort to measure stellar activity in solar-type stars. Many of these stars are in planet search programs (Nidever et al., 2002), and most now have high-quality radial velocities and metallicities. 1 The spectral type system is an astronomical shorthand that provides a basic characterization of individual stars. For historical reasons, the spectral type sequence is OBAFGKMLT (where L and T were added very recently), and runs from hot, high-mass stars to cool, sub-stellar mass brown dwarfs. The Sun is type G2. Confidential Page 12 2/12/2016 On the other hand, the Hipparcos sample included only a modest number of K dwarfs because only the very nearest were bright enough. However, analyses indicate that this sample of Hipparcos K dwarfs is effectively complete within 25 parsecs (Reid, Gizis & Hawley, 2002). Finally, nearby M and L dwarfs are the targets of an on-going campaign, the ML-NStars survey (Reid & Cruz, 2001; Cruz et al, 2003), based on near-infrared data from the 2-Micron All-Sky Survey (2MASS). So far, results are available for approximately half the sky (Reid et al., 2004), with follow-up observations underway for the remainder. The combination of these three families results in a total of ~4,350 stellar systems, sufficiently small numbers to allow detailed consideration of each source, but sufficiently large to provide reliable statistics. About 30% of the stars with known planetary-mass companions lie beyond these distance limits. This is only a small number of objects, and so we will supplement the Solar Neighborhood sample with data for all of those stars, regardless of distance. While selection effects may bias planet detection in these more distant stars, those effects should be apparent from comparison with the solar neighborhood sample; indeed, trends and biases among the exoplanet hosts are of intrinsic interest, potentially shedding light on the exoplanet formation process, and it is important to have the full context available. 2.3 Creating the Solar Neighborhood Habitability Census “..there are known knowns; there are things we know we know. We also know that there are known unknowns; that is to say we know there are some things we do not know. But there are also unknown unknowns – the ones we don’t know we don’t know.” (D. Rumsfeld, February 2002) 2.3.1 Building the database The SNH Census is designed to incorporate astrometric, photometric, spectroscopic, and multiplicity data for the nearest stars. In brief, we need to understand what we know about the stars in the Solar Neighborhood, how well we know it, and what we don’t know: we aim to maximize known knowns, identify known unknowns, and minimize the Rumsfeldian unknown unknowns. The information collected in the database will be divided conceptually into several categories. Identifiers, such as the Hipparcos or HD catalogue number are needed to enable crosscorrelations with other databases. Phase space data will describe the position and motion of the star with respect to the solar system barycenter. Right ascension, declination, parallax, proper motions, and heliocentric radial velocity are all examples of phase space data. Phase space data are useful for identifying kinematic groups. Classifications will describe morphological characteristics, such as spectral class and multiplicity. Photometry will compile apparent magnitudes in standard filters and quantities derived from those data. Examples include Johnson/Cousins UBVRI optical broadband magnitudes, EB-V color excess, 2MASS near-infrared JHK magnitudes, and mid-infrared excess flux above the predicted photospheric level. Physical properties will describe fundamental stellar properties, usually determined by detailed analysis. Confidential Page 13 2/12/2016 Effective temperature, gravity, rotation, and overall metallicities or individual elemental abundances are examples. Empirical properties will describe stellar measurements of observable characteristics that lack a fundamental interpretation. Observations of stellar activity fall into this category, since activity cannot be quantitatively linked to surface magnetic field properties. Derived quantities will be a function of other database columns. The function may involve interpolation in a table of model results (e.g. bolometric corrections or ages based on isochrone fitting). The derived quantities will also include parameters of relevance and use to our biologist colleagues, such as the flux of UV and X-ray radiation, and the HZ location. We will pay particular attention to assembling data on known companions and the circumstellar environments of the nearby stars. In particular, we will record constraints (including upper limits on potential companions) from precision radial velocity surveys, astrometric monitoring, direct imaging, and transit monitoring. We will record measurements and limits on extended emission at infrared and radio wavelengths. These factors are crucial to assessing the likelihood of finding a habitable planet around an individual star, yet no existing (or proposed) database comes close to providing all this information. We will not only compile and catalog such data, but our proposed database will highlight well-defined areas of doubt and uncertainty in our knowledge of planetary systems in the solar neighborhood. We will specifically target stars lacking crucial data in follow-up observing programs with ground-based and space-based observatories. Finally, we will couple the astronomical data with biological criteria to define habitability indices for each star. CoI Turnbull already has considerable experience in this area from her work on both HabCat (Turnbull & Tarter, 2003a) and the TPF target database. We anticipate including indices based on such factors as the presence and orbital characteristics of any companions, the circumstellar environment, the level of stellar activity, the metallicity of the parent star and the location of the habitable zone. We expect that both individual indices and the criteria themselves will evolve over the course of this project. Data collection and archive contents: Basic data, such as photometry, astrometry or metallicity, will be garnered from the literature or our own observations. In many cases, there are multiple measurements of standard parameters, such as (B-V) color. Under those circumstances we will use our considered judgment to select the most reliable value (or values). Where necessary, we will apply appropriate adjustments to different sets of measurements to give selfconsistent results; for example, using different chemical abundance indicators can lead to systematic offsets in the zeropoint and/or scale of stellar metallicities (Reid, 2002). Besides individual parameters, we will include relevant images and spectra in the SNHC database. In addition to our own optical and infrared spectra, we will incorporate archived data from ground-based and space missions, either directly or through links to the appropriate archives. We will also provide appropriate tools for combining multiwavelength datasets. Implementing the database: We will construct the SNH Census as a relational database, allowing high functionality and easy access. STScI uses this type of database, specifically SQL databases, to organize data associated with HST, JWST and particularly the Multimission Archive at STScI (MAST). We will achieve an efficient implementation of the SNH Census by adapting tools used to create and maintain existing STScI databases. We will ensure that the Confidential Page 14 2/12/2016 protocols are consistent with National Virtual Observatory (NVO) requirements, to ensure the widest access to our compendium of results. We plan the following schedule: During Year 1 we will compile the initial version of the database, populated with basic stellar data. We will implement the first set of habitability crtieria, modeled on Habcat, in Year 2, and release version 1.0 of the SNH Census to our NAI team members, and solicit their input; identify key missing data and, where necessary, initiate related observing projects. In Year 3 we will update and expand the Census; incorporate initial results from dynamical simulations (Section 3.3) in habitability criteria. We will release v2.0 of the SNHC to the NAI general community, and solicit their input. In Year 4 we expect to be able to include initial results from the Kepler mission, providing data on statistical properties of gas giant exoplanets, if not terrestrial planets. We will continue to acquire key missing data. In Year 5 we will release v3.0 of the SNH Census, with updated habitability criteria and a more complete census of the nearest stars. 2.3.2 Supplementary observing programs A crucial aspect of our proposed research is our intention to create the SNH Census as an active database, rather than simply capturing the sum of knowledge at one moment in time. We will identify significant defects in our knowledge of the nearest stars, and actively pursue their remedies. Even at this juncture, there are obvious gaps in our knowledge that we are addressing through either on-going observing programs, or programs that will be initiated in the near future. Close binary companions Close binary systems are generally unfavorable as hosts for habitable exoplanets, since stable planetary orbits generally lie well beyond the conventional habitable zone. Observationally, detecting close, low mass companions is challenging, and many nearby stars, particularly M dwarfs, have not yet been subjected to sufficient scrutiny to determine their true multiplicity. We envisage several observational programs to tackle this issue. Subaru high-contrast coronagraphic near-infrared survey of nearby stars: HiCIAO (High Contrast Coronagraphic Imager with Adaptive Optics) is a second-generation instrument currently under construction by the exoplanet research group at NAOJ (National Astronomical Observatory of Japan), led by Dr. M. Tamura, for use on the Subaru 8.2-meter telescope at Mauna Kea. HiCIAO will be used in conjunction with Subaru’s new 188 actuator AO system and laser guide star facility and with planned future “extreme AO” systems. HiCIAO will have multiple optical modules including the AO module, the warm coronagraph pupil plane module, the high contrast optics module and cold IR camera module. It will be a uniquely powerful high contrast imaging system that is expected to improve over the performance of all existing such systems by at least a factor of 10 and to be the first such system to combine polarization and spectral differential imaging modes. In addition, its modular designed allows ongoing improvements and development of new coronagraphic techniques, including experimentation with pupil plane mask designs. HiCIAO first light is currently planned for December 2006, and Confidential Page 15 2/12/2016 an extensive campaign of coronagraphic surveys will commence in 2007 and are expected to conclude in roughly 2011. We (CoIs Kasdin, Littman, Spergel, Turner and Vanderbei) will collaborate with Dr. Tamura and his group in both the experimental development of high contrast coronagraphy using HiCIAO and on extensive survey observations of nearby stars deemed to be of interest for exoplanet studies. In particular, the Princeton coronagraph group will design and construct pupil plane masks for use in an appropriate HiCIAO front-end module, and NIR high contrast images will be obtained of suitable targets. The latter observations will aim to discover and characterize faint companions and/or low surface brightness circumstellar material. Additional high resolution spectroscopic observations (using the APO Echelle, for example) and thermal infrared measurements using Spitzer and/or ASTRO-F will also be obtained. The results will be integrated into the Solar Neighborhood Habatibility Census. AO imaging of young nearby M dwarfs: Brown dwarfs and giant exoplanets fade and cool rapidly with time; young stellar systems therefore offer the best prospects for direct imaging of these very low-mass companions. M dwarfs have lower luminosities than solar-type stars, and therefore better contrast ratios for detection. Young M dwarfs have particularly active chromospheres and coronae, particularly from X-ray observations. As part of the ML-NStars survey, approximately 100 Pleiades-age M dwarfs have been identified, and those stars are being imaged using both conventional AO (on the Gemini North telescope) and the Spitzer midinfrared space telescope (programs led by PI Reid). The Gemini observations have the potential to detect 15 to 10 Jupiter-mass companions at Jovian separations (5-10 AU, where 1 AU is the Earth-Sun distance). Non-detections are equally significant, since they set upper limits on the frequency of such massive companions, and hence on their potential to de-stabilize lower-mass planetary systems. These systems will also be targeted in the future using HiCIAO and new highcontrast coronagraphic techniques (see Section 2.4.1). Exoplanets, stellar metallicities and ages Doppler planets are found with higher frequency around stars with super-solar metallicities (Fischer & Valenti, 2005). Detailed analyses of the high-resolution spectra used to search for planetary companions also provide important information on properties such as individual elemental abundances and stellar ages (Valenti & Fischer, 2005). Several hosts have abundance ratios consistent with membership of the Galactic thick disk, indicating that planet formation has occurred over the full lifetime of the disk (Reid, 2006). CoI Valenti is continuing analyses of stars observed in on-going radial-velocity planet surveys. 2.4 Exoplanet detection programs Even with over 130 planetary systems currently known, statistics are fairly sparse. Thus, identifying additional extrasolar planetary systems is a high priority. This section outlines planet detection programs that are being undertaken by members of the current NAI team. These include searches for transiting or resolved systems, which offer the potential for direct observations of the planetary companions, and therefore the opportunity to compare exoplanets with the Solar System. We are also pursuing microlensing surveys, which probe lower masses and different regions of the Galaxy. Confidential Page 16 2/12/2016 2.4.1 High-contrast coronagraphy and planet detection Direct imaging of exoplanets is an essential objective for both astrobiology and NASA’s Exploration Program. This includes the challenging task of detecting and characterizing Earth analogs via the Terrestrial Planet Finder missions (TPF-C and TPF-I) as well as detecting gas giants or low-mass brown dwarfs in Earth-like orbits about nearby stars. This latter task is less challenging (a Jovian analogue at 1 AU from a sunlike star has a contrast ratio of 10 -8, as compared to 10-10 for an Earth-analogue), but yet has profound implications for system habitability. NAI funding will both 1) allow us to continue our technology research into coronagraphic instrumentation techniques suitable for the extremely high-contrast needed for terrestrial planet detection from space and 2) allow us to expand our activities into ground based applications that will advance the state-of-the art for detection of low-mass companions and the resolution of the structure of dust and debris disks orbiting nearby stars. Princeton University is a leader in pupil plane coronagraphic technologies, and CoIs Kasdin, Littman, Vanderbei and Spergel have supported NASA research into high-contrast imaging for TPF since 2001. They have a fully operational high-contrast imaging laboratory where tests of coronagraphs and wavefront control systems are ongoing. The technology being developed at Princeton is critical for the support of future space observatories. In addition, we are also collaborating on programs and instrumentation for ground based 8-meter class telescopes, including mask support for the extreme AO work on Gemini (led by Lawrence Livermore National Laboratory) and in coronagraph technology for the Subaru infra-red survey. Figure 2.1: (Left) An image of the residual speckle in the dark region of the elliptical mask PSF after a 1 second exposure. (Middle) A cross-section of the experimental and theoretical PSF; black is the theoretical PSF, blue is the experiment, and red is the Airy function envelope. (Right) The experimental image overlaid with a simulation of the bright regions in the theoretical PSF that are blocked by a mask. Figure 2.1 shows an example of our current results. Using an elliptical mask, we modify the point-spread function (PSF) of an unresolved source (i.e. a star) to the shape mapped out by the bright regions in the right-hand figure. We can used a “bow-tie” mask to block that light, giving the left-hand image, which allows one to reach flux ratios exceeding 10 -5 at angular separations beyond 4/D (where D is the diameter of the telescope mirror). The data plotted are for monochromatic light, but we are achieving similar sensitivities with white light. This flux ratio is comparable to that of hot jupiters or T-type brown dwarf companions to solar-type stars. NAI-related activities NAI support provides an ideal opportunity for an in-depth study of the engineering of remote sensing and how it may enable new frontiers in extrasolar astrobiology and is a major part of our Confidential Page 17 2/12/2016 overall research program. We will continue to pursue both theoretical and experimental paths to understanding and mitigating the issues associated with high-contrast imaging from both space and the ground. We will apply our pupil amplitude profiling technology to as broad a spectrum of high-contrast imaging applications as possible in support of the search for life. Our effort will include continuing research into ultra-high contrast via pupil masks and wavefront control for TPF, moving the technology to a high technical readiness level in preparation for the future phase A opportunities. NAI funds will also support a student and faculty to examine optimized pupil masks and data processing for ground based telescopes, such as Subaru, advancing the technology to the point that we can propose construction of advanced instrumentation (using additional non-NAI funds) and use them to carry out observing programs of the circumstellar regions of nearby-stars. In support of future space coronagraph missions, such as JWST and TPF, we will continue our laboratory work in achieving ultra-high contrast via a combination of pupil masking and wavefront control. Recently ordered deformable mirrors are expected to arrive soon, and the first two years of NAI support for laboratory optical work in Princeton will be devoted to demonstrating the viability of control algorithms, as well as testing pupil mask designs. NAI support will is also allow us to initiate a technology research program intended to demonstrate the viability of pupil mask coronagraphs for ground based, high contrast imaging. This involves four major tasks: 1) design of shaped pupils compatible with telescopes containing central obstructions and spiders (this is eased by the lower contrast requirements of the ground observing program.), 2) Optimization of shaped pupils for spectroscopic and photometric characterization of already discovered companions, 3) Complete performance analysis of ground based planet imaging including full diffraction effects, atmospheric distortions, adaptive optics, and speckle reducing software, and 4) High speed, image plane based adaptive optics. All techniques and approaches will be tested and verified in our coronagraph laboratory at Princeton. Our proposed research in coronagraphic techniques has direct synergy with the construction of the Solar Neighborhood Habitability Census: some targets for the ground-based (and, eventually, space) coronagraphic instruments will be identified from analyses based on the SNH Census; conversely, the results of our observations will provide direct feedback to the Census, allowing refinement of habitability criteria for individual stars and/or classes of stars. 2.4.2 Transiting systems Planetary systems that transit the parent star are particularly important to astrobiology since these systems provide the only currently available method of studying exoplanet dimensions and probing the the atmospheric structure and composition. Following the initial discovery of HD 209458 (Charbonneau et al, 2002), a handful of other systems have been identified from photometric monitoring programs (e.g. TrES-1, HD 149026 and several systems from OGLE). All are hot Jupiters, with periods of less than 4 days. These systems provide vital information on the intrinsic properties of exoplanets, allowing direct comparison with the well studied gas giants in the Solar System. Both the XO project and the N2K survey are designed to add further systems to the rather short list available at the present time. Confidential Page 18 2/12/2016 Collaborator P. McCullough is leading the XO project (McCullough et al, 2005), an active observational program to discover additional hot-Jupiters that transit stars bright enough for detailed studies in the immediate future with existing telescopes such as HST, Spitzer, Keck, and ESO’s VLT, and later for future telescopes, such as JWST and SIM Planetquest. CoI Valenti is closely involved in this program. Finding these systems in a timely manner is important in order to make follow up observations with orbiting observatories; for example, Spitzer's expected life lasts until late 2008. XO employs a dedicated telescope on Haleakala, Maui, to monitor approximately 100,000 stars in a year, and is expected to discover two transiting hot Jupiters per year for the next three years. The N2K program (Fischer et al, 2005) is specifically designed to identify transiting hot Jupiters. These observations target 14,000 FGK main-sequence stars and subgiants with V<10.5 and distances within 110 parsecs of the Sun. CoI Valenti is also participating in this program, particularly in detailed analysis of the high resolution spectra obtained in the course of the radial velocity monitoring. The last few years have seen advances in radial velocity precision that, when coupled with intensive observations, have allowed detection of planets to 14 Earth Masses (Santos et al., 2004). Within the next decade, surveys are expected to probe the existence of planets down to a few Earth-masses for periods less than 10 days, and are likely to discover terrestrial-mass transiting planets. JWST may well offer the potential to provide atmospheric diagnostics for those planets. Finally, the Kepler mission is scheduled for launch in June 2008, and is designed to search for transiting terrestrial planet systems through high-accuracy (better than 2 x10-5) white light photometry of 105 solar-type stars within a 100 square degree low-galactic latitude field in Cygnus. Collaborator Gilliland is a CoI on the Kepler science team. Initial results from Kepler will become available in the course of our NAI program, and those data, which will include detections of gas giants, will provide invaluable data on planetary demographics. Observations of all these systems will provide valuable input data on planetary properties for the SNH Census. 2.4.3 OGLE, Microlensing and planets Microlensing surveys broaden the scope of planet detection to lower masses and different Galactic environments. OGLE (Optical Gravitational Lensing Experiment) is a long term collaborative project between Princeton and Warsaw University Observatories, using a 1.3 meter telescope and a 8K x 8K pixel camera located at the Las Campanas Observatory in Chile to carry out photometric monitoring campaigns of large numbers of stars in selected fields. OGLE has already discovered several Jupiter mass planets, using two distinctly different methods: gravitational microlensing and transits. OGLE has discovered five confirmed transiting exoplanets (cf. Schneider 2005). OGLE data have also led to the discovery of two Jovian planets by means of gravitational microlensing (Udalski et al. 2004; Bond et al. 2005), and a new paper reporting the discovery of a third such planet is ready for submission to Nature. This newly discovered exoplanet is much less massive than Jupiter and is located at several AU from the primary star, within its habitable zone. We Confidential Page 19 2/12/2016 emphasize that gravitational microlensing is the only technique currently available that can discover Earth mass planets orbiting in the habitable zone. Over the next several years OGLE will streamline its software in two ways. Photometric accuracy will be improved to bring it closer to the photon noise limit (Kruszewski and Semeniuk 2003, Tamuz et al. 2005). This will make it possible to detect shallower transits, and therefore smaller planets. The OGLE automated alert system will be streamlined to be sensitive to smaller anomalies in microlensing light curves (Udalski 2003). This will make it possible to detect lower mass planets by means of microlensing, and it will significantly increase the detectability of an Earth mass planet in the habitable zone. As part of this NAI core team, CoI Paczynski, whose initiated Galactic microlensing studies (Paczynski 1986) and who pioneered the idea of exoplanet detection via microlensing (Mao & Paczynski 1991), will work with Princeton graduate students in the continuation and improvement of the OGLE survey, with the goals both of discovering terrestrial mass exoplanets in stellar habitable zones and of ultimately understanding their population statistics. These results will provide direct feedback to the SNH Census on the potential frequency and semi-major axis distributions of terrestrial-mass planets. 2.5 Using the SNHC: enabled research programs Our purpose in constructing the SNH Census is to codify what we know about the stars in the Solar neighborhood, with specific regard to their potential for supporting habitable exoplanets. We will use that knowledge to focus follow-on research programs. Some of those programs will emerge in the course of compiling the SNH Census; others can be identified in advance. Here, we give three examples: Exoplanet statistics This is the most direct and immediate application of the SNH Census. Since our compilation will include upper limits on exoplanet detections from radial velocity, imaging and other surveys, we will identify the region of parameter space (mass, separation, apparent brightness) covered by existing observations on a star-by-star basis. We will use statistical techniques, such as Bayesian analysis (see Allen et al, 2005), to analyze the ensemble data and constrain the likely underlying properties of planetary systems. These analyses will be particularly valuable, since we will be working with a volume-complete sample. Selecting nearby stars for high-contrast coronagraphy: This program is directly synergistic between two key sections of our proposal, the SNH Census and developing advanced coronagraphic instrumentation. Simple geometry dictates that the nearest stars offer the best prospects for detecting resolved low-luminosity companions. We (PI Reid, CoIs Kasdin, Turnbull, Turner, Valenti and Vanderbei and Soderblom) will use the SNH Census to identify stellar systems offering the best potential for supporting habitable exoplanets, and will target those systems for observation with HiCIAO and other ground-based coronagraphs. The young, nearby M dwarfs that are already being scrutinized through conventional AO observations are clearly high priority targets. Initially, these observations are unlikely to be capable of directly detecting terrestrial exoplanets, but they have the potential to Confidential Page 20 2/12/2016 detect gas giants (or brown dwarfs). The orbital characteristics of those companions set strong dynamical constraints on the potential existence of habitable terrestrial worlds (see Section 3.3). Optimizing GO target selection for Kepler: Kepler is effectively a pointed mission rather than a survey, since only data for pre-selected sources within the 100-square degree area are saved. The targets will be chosen from the Kepler Input Catalogue, which will include optical (Sloan) and near-infrared (2MASS) photometry for 5 million stars and will be released publicly. Besides the main program, Kepler will provide the opportunity for Guest Observer programs targeting up to 3,025 stars at any given time, with the observations having typical durations from 3 months to a year. Thus, Kepler has the potential to observe ~50,000 additional sources over the 4 to 6 year lifetime of the mission. We will use the results from our analysis of the SNH Census to guide selection of field K and M dwarfs for a Kepler GO program to search for habitable exoplanet companions.The photometric data from the Kepler Input Catalogue will permit an initial selection of candidates. Chromospheric and coronal radiation have the potential to set significant constraints on the prevalence of habitable M dwarf exoplanets. This is an issue that we will explore through both astronomical observations (Section 3.4) and laboratory experiments (Section 4.3). We will use follow-up spectroscopy of photometrically-selected candidates to identify sources with activity levels most consistent with the requirements for habitability. 2.6 A census with a future A "census" may suggest a one-time enumeration, but our Solar Neighborhood Habitability Census will not only be maintained and updated, but will also grow and develop. Maintenance will keep the database functional, and updating keeps the information in the database current, but it is the augmentations to the Census that will add to its value over the course of our proposed program and beyond. As we noted above, the Census will be more than an astronomer's tool and will be adapted to guide and inform the work of biologists too. That, in turn, will influence the astrophysical data compiled, and, especially, the derived quantities that are relevant to assessing stars as sites for habitable exoplanets. A vigorous and cogent collaboration of the kind the NAI supports will lead to a database that is powerful and which has multiple uses. 2.7 Summary The principal tasks outlined in this section are: Develop the Solar Neighborhood Habitability Census as an investigative tool for the collection and analysis of astronomical and biological data relevant to the existence of habitable exoplanets in nearby stars. Initiate and support surveys for exoplanets; in particular, develop advanced techniques for high-contrast coronagraphic imaging with ground-based telescopes and in future space missions. Confidential Page 21 2/12/2016 3. What are the intrinsic and extrinsic environments of exoplanets? 3.1 Introduction Characterizing planetary properties is a key part of the Goal #1 of the Astrobiology Roadmap. Planetary environments and habitability depend on those properties, notably the atmosphere, and on the external conditions imposed by the properties of the central star and the planet’s orbit. The latter factors are relatively straightforward to estimate for individual exoplanetary systems. For example, Figure 3.1 (from Reid et al, 2006) shows the ambient surface temperatures experienced by an exact Earth analogue if it were a satellite of a known exoplanets. Many of those planets are on elliptical orbits, leading to the substantial temperature variations illustrated in Figure 3.1; those variations alone make it likely that only a handful of such hypothetical systems could support life. Similarly, massive gas giants have consequences for the existence of as-yet undetected terrestrial planets in stable orbits within the conventional habitable zone, while stellar activity can hamper the development of life on ill-placed exoplanet companions. These issues are explored in Sections 3.3 and 3.4. Figure 3.1 Surface temperatures experienced by an Earth-analogue terrestrial satellite of the known gas-giant exoplanets; solid red points identify systems where the primary star has evolved beyond the main sequence, and the shaded region marks the approximate temperature limits for known terrestrial life. Planetary atmospheres affect habitability. Present-day models are growing in sophistication, but still lack strong observational constraints. One of our research goals is to obtain and interpret new observations of planetary atmospheres and exospheres. At present, there are only limited options for direct observations of exoplanet, notably spectral signatures during transits. Such systems also allow measurement of planetary mass and radius, constraining structural models. Confidential Page 22 2/12/2016 3.2 Planetary atmospheres Existing planet searches have established that in the Solar Neighborhood approximately 1 star in 200 has a “hot Jupiter” companion. Simple geometrical arguments imply that only ~1 in 2000 has a transiting planet (Fischer et al, 2005). Spectroscopic monitoring of ~4000 stars has yielded only two transiting planets (HD 209458b and HD 149026b), and extensive photometric searches for transits (mostly among much fainter stars) have so far revealed only another half dozen (see section 2.4.2). Statistically, we anticipate that another dozen planets that transit bright (V<10) stars await discovery. Members of our team will attempt to characterize the atmospheres of these planets using a variety of techniques as they are discovered. 3.2.1 Space-based observations of exoplanet atmospheres Planets that transit bright stars provide an important astrophysical laboratory for studying planetary atmospheres. Empirical studies of exoplanet atmospheres can utilize two transitrelated techniques: (1) searches for absorption of starlight by the annulus of planetary atmosphere as the planet transits in front of the star (e.g. Na in HD 209458, Charbonneau et al, 2002) and (2) searches for diminution of reflected starlight or planetary emission as the planet passes behind the star (Charbonneau et al, 2005). In both cases, differential measurements compare flux in and out of eclipse, which typically lasts a couple of hours. Only bright stars with transiting planets can be measured precisely enough to study the planetary radius, atmospheric composition (Charbonneau et al., 2002, Vidal-Madjar et al., 2004), exosphere extent (VidalMadjar et al., 2003), and infrared emission (Charbonneau et al., 2005; Deming et al., 2005). Significant amounts of atomic hydrogen may fill the Roche lobe around a “hot Jupiter”, forming an exosphere that covers 10-20% of the stellar surface during transit. This exosphere absorbs stellar Ly- flux during transit, providing a measure of evaporation, which can be significant on cosmological timescales. Only HST can currently measure this key far-UV diagnostic. Initially, the Space Telescope Imaging Spectrograph (STIS) was used to study absorption of starlight by hydrogen, carbon, oxygen, and sodium in the atmosphere and exosphere of HD 209458. During multiple transits of HD 209458b, Vidal-Madjar et al. (2003) detected a 154% reduction in stellar Ly flux, which they attributed to absorption by the planetary exosphere. The absorption was stronger in the blue wing of Ly, suggesting outward acceleration of evaporated material due to stellar radiation pressure. While UV observations with STIS are no longer possible, similar data can be obtained using prisms on the Advanced Camera for Surveys (ACS). We have an accepted HST program (led by CoI Valenti) to directly detect the atmosphere and extended envelope (exosphere) of the recently discovered transiting planet HD 149026b. These studies are not directly funded by this proposal, but the results constrain planet formation scenarios, and feed directly into the SNH Census. . 3.2.2 Ground-based observations of transiting systems Observations from the ground are also possible. As part of this proposal, we (CoI Turner, working with Princeton graduate students and collaborators Y. Suto, J. Winn & T. Yamada) will exploit the high dispersion and extremely high signal-to-noise spectroscopy obtained with large ground based telescopes to characterize exoplanet systems via a variety of techniques. Extensive data have already been obtained with the Subaru 8.2-meter telescope’s High Dispersion Confidential Page 23 2/12/2016 Spectrograph and with Apache Point Observatory’s 3.5-meter echelle spectrograph for multiple “hot Jupiter” systems, particularly HD 209458b; further observations with these facilities are planned and observing time using the Keck 10-meter telescopes’ HIRES spectrograph has been requested. Integrations of tens of hours with an 8-meter class telescope on stars brighter than 10th magnitude at resolutions of nearly 105 yield signal-to-noise ratios of 103-104 per spectral resolution element with elaborate and careful data reduction procedures (Winn et al. 2004), and thus allow detection of small perturbations of the dominant stellar light due to the presence of the exoplanet. Such detectable perturbations include the exoplanet analogy (Queloz et al. 2000, Ohta, Taruya & Suto 2005) of the Rossiter (1924) - McLaughlin (1924) binary star effect by which the misalignment between the star’s spin angular momentum vector and the planet’s orbital angular momentum can be measured (from line profile variations) for transiting systems. A determination of this misalignment angle for HD209458b with unprecedented precision, 4.5 +/1.5 degrees, has recently been achieved by our group (Winn et al. 2005), and we plan to attempt similar measurements for sufficiently bright transiting “hot Jupiter” systems, starting with TrES1 and HD189733. The distribution of this angle is a fundamental constraint on models of planet formation and “migration” as well as containing information about the perturbation history of the system subsequent to planet formation. Furthermore, if it could be established that this angle is always, or nearly always, small (as it is in the two known cases of the Solar System and HD209458b), it would provide a powerful aid to exoplanet detection and characterization studies (see the review by Hale 1994 for details) and to targeting strategies for future exoplanet-related space missions such as SIM and TPF. The same data set can be used to search for the signatures of the evaporating atmosphere, the exosphere, of a “hot Jupiter” exoplanet via transmission spectroscopy in the manner of VidalMadjar et al.’s (2003) detection of Lyman-alpha absorption associated with HD209458b transits. Our group has already employed the data to place the strongest upper limits available to date on exosphere absorption in several important optical lines due to the exosphere of the same system (Winn et al. 2004, Narita et al. 2005). Finally, these data can be used to search for starlight scattered from a “hot Jupiter’s” atmosphere. This process will add a small component, proportional in strength to the planet’s albedo and surface area, to the total detected light of the system containing a Doppler shifted (typically be several 10s of km/s) copy of the stellar spectrum plus any spectral absorption features associated with the planet’s atmosphere (Leigh, Cameron & Guillot 2003). A detection of, or strong upper limits on, the strength of such a component of the system’s total light, would provide a powerful probe of the atmospheric properties of this novel class of planets and be a major contribution to their physical characterization. Half a dozen exoplanets well suited to studies of this type are currently known and more will be discovered in the near future. Our team will also analyze the data obtained in this project in an attempt to make the first direct detection of such scattered exoplanet light. 3.3 Planetary dynamics System habitability can be affected by dynamical interactions within planetary systems. Aspects of these issues are being addressed by CoI Tremaine and collaborator Lubow. Their work Confidential Page 24 2/12/2016 complements ongoing research by current NAI teams at CIW, the University of Washington and the Unversity of Arizona. 3.3.1 Dynamical evolution of planetary systems Most work on planet formation is focused on the first Myr or so after star formation, during which time planets are believed to form through a complex process involving four main stages: (1) dust settles out of the cooling protoplanetary gas disk; (2) the dust concentrated in the midplane of the disk coalesces into km-sized bodies (planetesimals) through sticking processes or gravitational instabilities; (3) the planetesimals grow by collisions into planetary embryos; (4) the growth of the embryos stalls once they exhaust the material in their feeding zones, until the cumulative effects of long-range gravitational interactions pump up their eccentricities to the point where collisions and further growth into planets can occur (e.g., Lissauer 1993). We may call this stage of planet formation “Phase I”. The evolution of planetary systems during Phase I is difficult to disentangle, since the processes are complex and observations are scarce. Collaborator Lubow is investigating the possible role of disk/planet torques might play in defining the radial number distribution and eccentricity distribution of exoplanet systems. Previous theoretical studies of migration have focused on the properties of the so-called Lindblad resonances, where disk material sweeps through the gravitational force field of the planet (Tanaka, Takeuchi, and Ward 2002 and references therein). Corotational resonances, which have received less attention, are a potentially stronger influence on early orbital evolution. Lubow, in collaboration with D'Angelo, Bate (Exeter) and Ogilvie (Cambridge), has been analyzing the role of corotational resonances in disk/planet interactions. They find that fast exoplanet migration occurs under certain conditions, and that material trapped by the planet as it migrates can accelerate the migration. These interactions can also cause the excitation of planetary eccentricity if the planet opens a gap in the disk material. The results from these studies provide constraints on the potential existence of as-yet undetected low-mass exoplanets in known planetary systems. Following Phase I there is a second, much longer interval (5-10 Gyr; we call this “Phase II”) in which the planets interact only through gravitational forces. During this stage planets can be ejected into interstellar space, collide with the central star or other planets, be captured into resonances via three-body interactions, etc. The importance of Phase II in determining the properties of observed planetary systems is unknown, but a possible indication of its significance is the observation that the outer solar system is “full”, in the sense that no other planet on a stable orbit could be squeezed in between Jupiter and Neptune (Holman & Wisdom 1993); either this is a coincidence or, more likely, there were once additional planets in the outer solar system that were ejected during Phase II. We (CoI Tremaine, working with Princeton graduate student M. Juric) have begun using numerical models to investigate Phase II. The numerical integrations will be based on the “Mercury” code (Chambers 1999), with extensive modifications to handle highly eccentric orbits accurately and efficiently. As part of this NAI proposal, we will run the integrations on the Astrophysical Sciences cluster computer (currently about 200 nodes with 3 GHz clock speed). A major challenge to these studies is that the initial conditions are unknown; consequently, we need Confidential Page 25 2/12/2016 to consider a wide range of starting points. We expect to be able to run a suite of simulations, typically 1000 planetary systems followed for 100 Myr, in about a week of wall-clock time. Preliminary experiments suggest that: (1) most systems lose at least half their planets in Phase II; (2) the surviving planets acquire a distribution of eccentricities similar to that seen in the sample of known extrasolar planets; (3) the mean eccentricity in a system is anti-correlated with the number of planets. The last of these results, in particular, suggests that planetary systems can be arranged along a sequence: at one end are the commonest systems, with 1 – 3 planets having relatively large eccentricities, while at the other are rarer systems having more planets with smaller eccentricities. The observed extrasolar planetary systems are at one end of this sequence, and the solar system at the other. Not only will these simple experiments explore an important but neglected component of the puzzle of planet formation, but if – as we expect – the Phase II evolutionary effects are strong, then at least some properties of observed planetary systems may be deduced without a complete understanding of the far more difficult Phase I. 3.3.2 Small planets Since planets are believed to be formed by the accretion of vast numbers of small planetesimals, and since this process stalls when the growing planets clear out their feeding zones, at masses ≈1024 g (Rafikov 2001), it is natural to wonder whether there are planetary systems in which this process did not proceed as far as in our own system. Could there be systems containing hundreds of lunar-mass bodies, or thousands of Ceres-mass bodies? Could these perhaps be far more common than “terrestrial” and “giant” planets? As part of this proposal, CoI Tremaine, working with Princeton graduate students, will investigate the structure and dynamics of systems containing many small planets (in which the overall mass of non-volatile elements in the planetary system is similar to what is observed in the Solar System and extrasolar planetary systems, i.e., 10 – 100 Earth masses). In particular, we shall ask: Under what circumstances can such systems survive to the present time, against gravitational scattering and physical collisions? Under what circumstances could the growth of planetesimals into planets stall? How could we detect small-planet systems? The most promising possibility is to use gravitational microlensing, because the surface density in the disk determines the overall cross-section for microlensing by a planetary system, while the planet mass affects only the duration of individual microlensing events. What is the relation of small-planet systems to debris disks (Rieke et al. 2005)? 3.4 M dwarf exoplanet environments Solar-type stars (G, or sometimes F-K, dwarfs) have been the main focus of exoplanet investigations over the last decade or more. These stars, however, are a minority constituent of the Galactic disk: 80% of local stars are M dwarfs, with masses below 0.5 M⊙. In these systems the conventional habitable zone lies at smaller separations; indeed, Kasting et al (1993) estimated that planets in circular orbits in the habitable zone (HZ) of solar-age M dwarfs are likely to be Confidential Page 26 2/12/2016 tidally locked, with one hemisphere illuminated constantly by the central star and the other perpetually in the dark. Synchronously rotating exoplanets seem inhospitable, yet Haberle et al (1994) hypothesised that an atmosphere of more than 0.1 bar of CO2 could provide sufficient heat transport to prevent freezing on the unilluminated hemisphere. (Planets on elliptical orbits that intersect the HZ are not synchronously locked, although they undergo long-term cyclical variations comparable to those modeled in the experiments discussed in Section 4.2.) More detailed models of synchronously rotating planets (Joshi et al, 1997; Joshi, 2003) support Haberle et al’s conclusions. There has been growing interest in habitable M-dwarf exoplanets; in particular, the SETI NAI team recently convened a series of workshops to investigate relevant issues (PI Reid and CoIs Chyba and Turnbull are involved in these studies). An issue of particular interest for M dwarf exoplanets, and directly relevant to the experiments outlined in Section 4.3, is the role played by high-energy chomospheric and coronal radiation, generated by the stellar magnetic fields. M dwarfs are proportionately more active than solartype stars, and can emit 10% of their total energy as ultraviolet and X-rays. Many M dwarfs have the same absolute X-ray luminosity as the Sun; since the conventional HZ lies at ~0.3 AU, an M dwarf exoplanet is subject to X-ray and particle fluxes ten times that of Earth. Moreover, during flares, which occur more frequently than on the Sun, the high-energy flux can increase a further hundredfold. This non-thermal radiation might provide an additional source of heating for planetary atmospheres, but could also lead to disadvantageous mutations in living organisms. To what extent does this radiation environment affect planetary habitability, and how do these emissions change over time? M dwarf activity has two components: an approximately constant flux from the quiescent chromosphere/corona; and short-lived (minutes to ~few hours) flares, where the ultraviolet and X-ray fluxes can increase by factors of 102 to 104 over inactive levels. The NAI JPL/Caltech Virtual Planet Laboratory team is currently modeling some effects due to quiescent activity for selected M dwarfs; PI Reid is a CoI on a related Hubble Space Telescope proposal (PI: L. Walkowicz, U. Washington), using the Advanced Camera for Surveys (ACS) to obtain low-resolution ultraviolet spectra of nearby M dwarfs, measuring the range of quiescent activity as a function of spectral type. Walkowicz is also measuring H and Ca II H&K emission-line strengths for these stars, providing coss-calibration of multi-wavelength activity indicators. Activity in M dwarfs is likely to vary over a wide range of timescales: 1. there is a long-term decline in activity with increasing age; 2. flares decrease in frequency with age, although there are no reliable determinations of the time dependence (does it scale with the quiescent flux?) or the variation in the peak flux; 3. there may be short-term (few year) cyclical variations in activity, comparable to the Solar cycle. Superimposed on all these variations is a general trend: activity declines at a slower rate with decreasing mass. The long term decline in quiescent activity is best studied through observations of stars in open clusters of various ages. Hawley et al (1999) completed an initial analysis, based on observations Confidential Page 27 2/12/2016 of M dwarfs in 6 clusters with ages between 30 million years (IC 2602) and 5 Gyrs (M67). The sample does not include any clusters with ages between the Hyades (500 Myrs) and M67. The activity level was gauged by determing the spectral type where H emission becomes apparent. We propose to extend this age-activity calibration by obtaining optical spectra of M dwarfs in open clusters with ages between 1 and 5 Gyrs.We aim to use multi-object spectrographs on the Gemini telescopes, and the KPNO and CTIO 4-meter telescopes for these observations. Besides long-term changes that occur as stars age, shorter-term activity cycles exist. Those cycles are well-established for Sun-like stars but are little studied at later spectral types. The possible existence of cyclical variations in M dwarf activity levels is currently being addressed by CoI Valenti, who is using SMARTS-consortium time to monitor Ca II H&K emission in selected neaby M dwarfs. The current observations span two years, and this program will be continued in the near future. Finally, while M stars are famous for their high-intensity flares, quantitative measurements of the variation in flare activity with age remain sparse. While it seems clear that flare frequency must decline with age, along with quiescent activity, observations tend to concentrate on younger, more active stars. We propose to address this issue by photometrically monitoring M dwarf stars in open clusters, concentrating on systems with ages between 1 and 5 Gyrs. These observations can be made in tandem with searches for planetary transits, with the important proviso that data are taken at blue wavelengths, to provide maximum sensitivity to flares. The observations will be obtained and analysed by Reid, Turnbull, Soderblom and collaborator Sahu. We will use the CTIO telescopes that are part of the SMARTS consortium to study southern clusters in the appropriate age range. As results are obtained for these closely related M dwarf projects, the astronomer CoI’s Valenti, Reid and Turnbull will work with the extremophile CoI’s Robb, DasSarma and Chen to understand how microorganisms can survive, and even flourish, under M dwarf-like radiation environments. 3.5 Summary The principal tasks outlined in this section are: Pursue the characterization of exoplanet atmospheres through ground-based and spacebased spectroscopy of transiting exoplanets Model the dynamical formation and evolution of exoplanetary systems Determine the range and time-variability of the high-energy radiation environments experienced by M dwarf exoplanets Confidential Page 28 2/12/2016 4. Can the exoplanets support life? 4.1 Introduction Astrophysical environments span a range of harsh conditions. Within the Solar System, average surface temperatures range from 300°C for Mercury to -230°C for Pluto, with greater extremes occurring locally and seasonally; many known exoplanets are subject to an even greater range of conditions (see Figure 3.1). Nonetheless, recent studies of diverse environments on Earth suggest that some forms of microbial life have adapted to a significantly wider range of conditions than previously thought possible (Faison, 2003). Besides temperature extremes, these include anaerobic conditions, denaturing and caustic chemical species, such as salts, hydrogen and hydroxide ions, heavy metals, etc., and damaging radiation and desiccating conditions (Rothschild and Mancinelli, 2001). Indeed, some microorganisms thrive in (and even require) these extreme conditions; such species have been dubbed “extremophiles”. Many are Archaea, members of life’s third domain, while most prior NAI studies have focused on Bacteria. We have selected a representative set of terrestrial microorganisms, including cold-adapted and radiationresistant species, that are particularly well suited for further investigation of life’s limits – a crucial aspect of Goal #5 in the Astrobiology Roadmap. The COMB team of researchers will target microbial life forms that likely evolved at an early stage in Earth’s history, and which will provide insight into possible life elsewhere in the Solar System and on exoplanetary systems (Table 4.1). The Archaea were recognized as a separate taxonomic group just over 25 years ago (Woese and Fox, 1977; Woese, 1987). They display relatively simple growth requirements and are thought to be evolutionary relics. Although their early history is unclear, studies suggest that Archaea are genomic chimeras or hybrids, containing features of information transfer systems similar to higher organisms, and with metabolic capabilities similarities to Bacteria. Halophilic Archaea (e.g. Halobacterium species) contain a simple retinal-based photopigment which may have evolved even earlier than chlorophyll-based photosynthesis, a hypothesis based on the complementarity of the spectra of chlorophyll and retinal-based pigments (DasSarma, 2004). Methanogenic Archaea can grow in the most minimal components, such as H2 and CO2 or CO. Methanogens are capable of growth in an extremely wide range of temperatures, from below 0oC to a high of 110oC. Some of the earliest fossil records of life on Earth are found in stromatolites, formed by microbial communities. We will therefore also investigate species in modern microbial mat or biofilm communities, including those present in saline, hypersaline, and anxoic environments (Cohen and Rosenberg, 1989). Significantly, the photosynthetic activities of such populations contributed significantly to changing Earth’s atmosphere from reducing to oxidizing, altering the course of evolution and leading to the development of higher organisms. For example, photosynthetic cyanobacteria, such as Synechococcus spp., contain chlorophyll-based photopigments similar to plants and may be ideal as founding organisms (Schopf 1993). They have the capability to grow on minimal chemical components, utilizing the energy of sunlight and the ability to fix CO2 (the "dark reaction" of photosynthesis). Some purple bacteria, e.g. Rhodospirillum spp., may grow photosynthetically within anaerobic zones, utilizing H2S as an electron donor for CO2 fixation, and also autotrophically, via CO oxidation. Confidential Page 29 2/12/2016 In selecting individual species for study, our top priorities are representative cold-adapted halophilic and methanogenic microorganisms isolated from Antartica (see Section 4.2) which we have studied over the past two years in an inter-institute collaboration (STSCI and COMB). These have been compared to mesophilic relatives through comparative genomic, biochemistry and physiological approaches (Table 4.1). The mesophiles are model organisms which have been completely genetically sequenced, are transformable, and have plasmid vectors and other genetic tools for use. Our first report on the finding of methanogenesis and biofilm formation at subzero temperatures has been submitted recently to Astrobiology (see below; Reid et al. 2006). Table 4.1. Microbial species for probing life’s limits to cold temperature. Organism type Experimental Model Species Halophilic Archaea Halorubrum lacusprofundi Halobacterium NRC-1 Methanogenium Methanogenic frigidum Archaea or Terrestrial Habitats Characteristics relevant to astrobiology Hypersaline Extremely halophilic, radiation resistant, Antarctic lake psychotroph, growth at < 0 oC sp. Great Salt Lake, Extremely halophilic, radiation resistant, solar salterns mesophile, genetically tractable model Saline, anaerobic Halotolerant, strict anaerobe, Antarctic lake methanogenic psychrophile to < 0 oC, autotrophic growth with H2 and CO2 Methanococcoides Saline, anaerobic Halotolerant, strict anaerobe, burtonii Antarctic lake methanogenic psychtolerant to -2 oC, growth with simple methylated molecules Methanosarcina Saline, Halotolerant, strict anaerobe, mesophile, acetivorans marine sediments genetically tractable model Marine sediments Halotolerant, omniadaptive growth via Green nonsulfur Rhodospirillum rubrum respiration, fermentation, photosynthesis, Bacteria photoautotrophy, autotrophic growth with CO, tractable model, 5-10oC Synechococcus spp. Estuarine, coastal, Photosynthetic, autotrophic with CO2, Cyanobacteria CB0101, CB0205 open ocean radiation resistant, highly diverse and ancient microbes, tractable, growth limits not yet tested In addition to studying the mechanisms of cold-adaptation of currently cultured organisms, we will also prioritize new microbial enrichments from Arctic, subarctic, and Antarctic Mars analog sites by working closely with our collaborators on this proposal and with other NAI teams. This work is likely to expand our breadth of understanding of species diversity and metabolism operating in the cold. Finally, we will also study radiation-tolerance of microorganisms, which is likely to serve as a significant advantage for survival on planets subjected to high radiation intensities, including Mars, and for possible dispersal of life in our galaxy. These studies will be particularly relevant from the standpoint of planetary protection, especially for Mars. In summary, our strategy will permit us to probe the limits of life on Earth as well as define the relevant physiological and genomic mechanisms operating under stress. These priorities include selected collaborations with scientists at other NAI nodes which will complement and extend our work and enhance the overall NAI effort in extremophile biology. 4.2 Life in the Cold From the human perspective, Earth’s environment is clement. However, 80% of the biosphere never experiences temperatures higher than 5oC. In the Solar System, Mars and Europa, the two Confidential Page 30 2/12/2016 best candidates for life, both experience average temperatures well below 5oC, as do many exoplanets (Figure 3.1). Cold environments are commonplace throughout the Galaxy. In addition, surface temperatures are not stable over time; extreme temperature variations occur on a diurnal and seasonal basis as well as with longer-term periodic or chaotic changes in orbital parameters (e.g., Mars’s obliquity). Therefore, thermal cycling events have a significant impact on predictions of survival. In our own Solar System, for example, freeze-thaw cycles in the Martian near-subsurface have been cited as one of the strongest challenges to the survival of terrestrial bacteria that may be brought to Mars on Earth spacecraft (National Research Council 2005). On Earth, Antarctica has one of the driest and coldest habitats and provides scientists with a diverse inventory of cold-loving (psychrophilic) and cold-tolerant (psychrotolerant) organisms. Such microorganisms represent highly specialized and frequently highly localized microbial biotypes, including those associated with ice-covered and permanently liquid lakes, glacial and sea ice, and a wide range of soils and rocks. They are characterized by one or more environmental extremes (including low temperature, wide temperature fluctuations, desiccation, hypersalinity, high periodic radiation fluxes, and low nutrient status). Several species of halophilic and methanogenic Archaea isolated from the Vestfold Hills, including Halorubrum lacusprofundi, Methanogenium frigidum Ace-2 and Methanococcoides burtonii, are cultured in our laboratories and serve as genetic and physiological experimental systems. These species serve as comparative partners to mesophiles from diverse environments (see Table 4.1). 4.2.1 Cold and salt: Psychrophilic halophiles The freezing temperature of pure water at terrestrial standard atmospheric pressure (101.3 kPa) is 0oC. By contrast, brine-saturated water can exist in liquid form below –20oC and other salts can depress the freezing point to –50oC or lower. A number of hypersaline lakes have been studied from Antarctica, including Dry Valley lakes (Lake Bonney, Lake Vanda, and Don Juan Pond) and Deep Lake (in the Vestfold Hills) (Javor, 1989). Some lakes contain greater than 25 % total dissolved salts. Several extraterrestrial bodies have saline environments, notably Mars and Europa, so the study of salt-tolerant microbes is highly relevant to the aims of our project. Halorubrum lacusprofundi is a halophilic Antarctic species isolated from Deep Lake (Franzmann et al., 1988). This is an aerobic species reported to be capable of growing at 2-4oC, although recently shown to grow at -1 oC in our COMB-STSCI collaboration (Reid et al. 2006). Thus, it can survive and divide at sub-zero temperatures in essentially saturating concentrations of NaCl. Phylogenetically, H. lacusprofundi is a member of the halophilic Archaea, and contains transcription and DNA replication systems similar to higher organisms. This archaeon is highly pigmented and contains the light-driven proton pump, bacteriorhodopsin, used for phototrophic growth. A draft sequence of the genome of H. lacusprofundi has been determined in the DasSarma laboratory and is currently being completed through DOE’s Community Sequencing Program at the Joint Genome Institute (JGI). DasSarma and collaborator R. Cavicchioli, University of New South Wales, are CoPIs on this project and the complete sequence will be available to us soon, six months before public release in late 2006. A similar but mesophilic haloarchaeon with a growth range of 15-55oC is Halobacterium sp. NRC-1 (DasSarma, 2004). This microbe is found in the Great Salt Lake and other evaporitic Confidential Page 31 2/12/2016 hypersaline environments that are characterized by salinity levels of 3-5 M NaCl and intense solar radiation. Halobacterium sp. NRC-1 is a model organism studied for 20 years in the DasSarma laboratory, which is genetically tractable and for which a host of post genomic methods, such as directed gene knockouts, DNA microarrays and proteomics, have been developed. The response of Halobacterium to changes in salinity, temperature, nutrients, light, radiation and oxygen have been studied (Müller and DasSarma, 2005; McCready et al. 2005) and will provide an exceptionally good mesophilic relative for comparison to H. lacusprofundi. Specific aims: (1) To determine the low temperature limits to growth of the psychrophilic halophile, H. lacusprofundi in comparison to its mesophilic relative, Halobacterium sp. NRC-1. (2) To examine the mechanisms for survival of haloarchaea to environmental dynamics (e.g. freeze-thaw cycles, desiccation-hydration cycles) through physiological and genomic approaches. Experimental plans: The effect of temperature on growth dynamics will be assessed through growth in a variety of media (DasSarma and Fleischmann, 1995), experiments already initiated by CoI DasSarma and collaborator J. Coker at COMB. Initially, nutrient content will be increased to the maximum permissible by solubility, which should enhance growth and depress freezing points. Alternatively, other salts and water miscible organic solvents will also be tested. Finally, a combination of nutrients, salts, and solvents will be used to approach the low temperature growth limit. We will use liberal criteria for assaying growth, given the time scales appropriate to growth on exoplanets and our recent observation that cultures may adapt to colder temperatures over time (Reid et al, 2006). We will monitor cultures over a 3-year time period. Once the lower limit of growth is determined, an additional goal will be to determine the extent of survival at temperatures sub-inhibitory for growth (Franzmann et al. 1988). We wish to explore whether organisms may survive for extended periods of time when temperatures are too low for growth, allowing return to active growth when temperatures and other conditions permit. This is an important goal given the orbital ellipticity of many exoplanets, which may result in long winter seasonal cycles. What temperature extremes are tolerable without complete loss of viability? How long do cells survive after freezing and how many cycles of freeze-thaw may be tolerated? A multiyear time-line will be followed, allowing a thorough comparative study of the survival of these haloarchaea. Analysis of both psychrophile and mesophile will allow us to determine if the former has devised specific mechanisms to permit survival in quiescent state for an extended period of time. In addition to establishing the effect of freeze-thaw, we also plan to carry out desiccationhydration cycles to determine the capability of the halophiles to withstand such stresses (Landis, 2001). Although halophiles have been reported from ancient salt deposits (Vreeland et al. 2000), their survival over multiple rounds of desiccation and rehydration has not been studied (Kotteman et al. 2005). No spores have ever been observed in this class of microorganisms, so it is likely that novel strategies have been devised to allow survival in arid environments. Once the parameters of survival and death have been defined, we plan to address the underlying mechanisms through a genomic approach. A whole genome oligonucleotide microarray has already been used successfully for the mesophile (Müller and DasSarma, 2005; McCready et al. Confidential Page 32 2/12/2016 2005) and a similar microarray will be constructed for the psychrophile (see Section 4.2.4.1). Microarrays provide a powerful tool that, in combination with physiological approaches described above, will address the biological responses relevant to astrobiological conditions. The regulated genes will be compared between the two strains and will provide the basis for understanding the mechanism of adaptation, besides suggesting possible gene transfer experiments (see Section 4.2.4.2). 4.2.2 Cold and anoxia: Psychrophilic methanogens Psychrophilic methanogens represent life forms that exist in perennially cold, anoxic conditions with minimal nutrient requirements. Methanogens use hydrogen and hydrogen rich compounds and carbon dioxide to produce energy and release methane. Knowledge of the adaptation mechanisms for growth and survival of these microorganisms will provide insight into the extreme limits of life that might arise in cold exoplanets with oxygen-free atmospheres. Genome sequencing of the psychrophilic methanogen, Methanogenium frigidum (theoretical Tmin –1.8oC) and the psychrotolerant methanogen, Methanococcoides burtonii (Tmin 1.7 oC) was recently initiated. These archaeal species were isolated from a perennially anoxic zone of Ace Lake in Antarctica. Constant temperatures of 1 to 2oC, salinity of up to 4.3 % and high concentrations of methane and H2S further characterize this region of the lake. The ability to grow at low temperatures, in the absence of oxygen and with only hydrogen or simple organic compounds, make these microorganisms particularly relevant for assessing habitability, since these conditions are associated with the early formation of the Earth’s biosphere. This study combines physiological and genomic data to identify survival characteristics that can constrain the diversity of microbial species expected in cold exoplanets. Completing the M. frigidum and M. burtonii genome sequences will expand our understanding of the organismal and metabolic biocomplexity of cold-adapted Archaea, and provide the reference for comparing mechanisms of archaeal cold-adaptation with those in Bacteria and Eukarya. Completed genomic sequences combined with proteomic and genetic approaches will be used to characterize the absolute limits of adaptation and identify cold-adaptation mechanisms. A Specific aims: To determine the lowest temperatures that support growth and/or methanogenesis by these microorganisms, the longevity of these microbes at temperatures below the growth range, and the range of temperature fluctuations (freeze-thaw) under which they can survive. To elucidate mechanisms by which survival under these conditions takes place, we will investigate the physiological and genetic factors that limit growth at the lowest temperatures. Experimental plans: To date there have been only two reports (Franzmann et al, 1992; 1997) describing the physiological characteristics of M. burtonii and M. frigidum. Although both suggested that the archaeal species grew sub-optimally at their in situ temperature in the laboratory, CoI Sowers showed that M. burtonii growth and methanogenesis occur at sub zero temperatures (Reid et al, 2006). We will continue adaptation studies with M. burtonii and initiate adaptation studies with M. frigidum to determine the lowest temperature for both growth (cell doubling) and methanogenesis (physiological activity). Both species will be maintained and monitored at –15 to 0 oC using refrigerated water bath shakers containing antifreeze. As the marine media for both species freezes at -5 oC we will add a cryoprotectant (such as glycerol) to Confidential Page 33 2/12/2016 prevent freezing. We will also test the ability of frozen cultures to recover after storage in medium without cryoprotectant for different periods of time and we will also test the efficiency of recovery for cultures after repeated freeze-thaw cycles. The ability of these species to maintain metabolic activity at subfreezing temperature will be determined by monitoring methanogenesis over time in frozen cell suspensions. Overall the experiments will define the limits for (1) growth, (2) metabolic activity and (3) recovery after stasis. 4.2.3 Cold and Carbon Monoxide The simplest autotrophic growth requirements on record are provided by the Bacteria and Archaea that grow in the dark under anaerobic conditions with CO as the sole source of carbon and energy. This is accomplished via the gas-shift reaction, CO + H2O ↔ CO2 + 2 e- + 2 H+. The key enzyme in this process is carbon monoxide dehydrogenase (CODH) that catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster (Drennan et al. 2001) using this 2-electron, hydrogen producing process. We will study , Rhodospirillum rubrum, capable of growth with CO alone and at temperatures as low as 5-10 oC. We will contrast this species with another CO oxidizer, Carboxydothermus hydrogenoformans. These strains are distinct in their taxonomy and physiology: R. rubrum is a mesophilic, Gram negative, purple nonsulfur bacterium that can grow aerobically or anaerobically. It is exceptionally versatile and has the ability to live through photosynthesis, cellular respiration, fermentation, or chemoautotrophic growth. In contrast, C. hydrogenoformans is an extreme thermophile, able to grow rapidly on high levels of CO and grows poorly on a very limited assortment of substrates. We recently completed the genome sequence of this low GC, Gram positive bacteriun, and found that it has five genetic loci encoding CODH, one of which is remarkably similar to the single cdh locus in R. rubrum (Wu et al, 2006) Anaerobic CO oxidation may be a model for microbial growth in primitive planetary atmospheres (Weiss et al, 2000). CO is the second most abundant molecule, after water, in the ice mantles of interstellar grains (Ehrenfreund, 1999, Charnly et al. 2001) and thus the study of terrestrial organisms that specialize in CO oxidation is astrobiologically relevant. The chosen representatives have a draft genome sequence from the JGI, in the case of R. rubrum, and a newly published, complete genome sequence, in the case of C. hydrogenoformans (Wu et al, in press). These will provide the basis for comparative studies on the thermodynamics of metabolic pathways providing energy and carbon fixation from CO oxidation. Specific aims: The free energy (G) of CO oxidation is highly temperature dependent, and we will carry out enrichment and isolation of new psychrophilic CO-oxidizing strains and compare their growth yields and mass balances with the mesophilic and thermophilic model organisms. Experimental plans: CoI Robb and collaborator A. Colman will determine the minimum growth temperature of R. rubrum in batch cultures and chemostats, and test a wide range of growth temperatures, with a range of partial pressures of CO in the headspace. Mass balances for the cultures will be determined by measurement of biomass produced and through gas chromatographic headspace analysis to determine CO consumed and H2 produced. We will determine the adaptive response of the CO dehydrogenase encoding loci in R. rubrum and C. hydrogenoformans by transcriptional profiling, using microarray analysis. Spotted DNA microarrays have been developed in collaboration with Idaho National Laboratory. We will Confidential Page 34 2/12/2016 enrich from samples in Mars analog sites for psychrotolerant and psychrophilic CO oxidizing cultures and isolate pure cultures, in collaboration with collaborators Cavicchioli, Onstott, Steele and Fogel. We may find obligate consortial partnerships between CO-oxidizing bacteria and methanogens, and will propagate these systems under CO headspaces and determine species composition by 16SrRNA sequencing. Our NSF funded Microbial Observatories site in the Uzon Caldera, Kamchatka, also has permafrost sampling sites. Recently several methanogens have been grown on CO as carbon and energy source, and we will attempt to enrich for psychrophilic methanogenic consortia as well as acetogens from cold sediments. We will also carry out fluorescent in situ hybridization (FISH) to reveal interspecies juxtaposition in the consortia. Isolates will be subject to mass balance determination to test their efficiency of CO utilization, methanogenesis, acetogenesis and hydrogen production. 4.2.4 Mechanisms of cold adaptation The low temperature growth capability of M. burtonii and H. lacusprofundi reflects the genomic composition of these highly adapted microorganisms. Since partial genome sequences are available, we have conducted an inventory of predicted proteins known to be important for coping with low temperatures. Strikingly, the genome of H. lacusprofundi contains 5 copies each of the two different cold shock protein (CSP) genes while the mesophilic Halobacterium sp. NRC-1 contains only one copy each (cspD1 and D2). In general, CSPs are involved in various cellular processes and believed to enable cells to adapt to low temperature (Thieringer et al. 1998). They can bind to single stranded DNA and RNA and have been suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures. Overproduction of CSPs leads to increased survival during periods of freezing (Wouters et al. 2000) and the presence of multiple copies of CSP genes in H. lacusprofundi suggests a molecular basis for the cold-adapted nature of this organism. Figure 4.1 Scanning electron micrographs of cell aggregates formed by H. lacusprofundi grown at 4 oC (A) and Methanococcoides burtonii grown at –2.5 oC (B). The bars represent 2 m. Comparative analysis of the incomplete genomes of the psychrotolerant M. burtonii and the psychrophilic M. frigidum, also isolated from Ace Lake, revealed several potential mechanisms (Saunders et al., 2003). A gene with high homology to the cold shock domain (CSD) protein cspA was detected in the genome of M. frigidum. In contrast cspA was not detected in M. burtonii, but two proteins were detected that contain the highly conserved CSD fold characteristic of CSPs. Another unique characteristic of the cold adapted methanogens is the Confidential Page 35 2/12/2016 amino acid composition of proteins, which generally increase exposure of hydrophobic residues and decrease charge. This modification was suggested to decrease the activation energy by increasing protein flexibility. Cell aggregation might be a common high temperature-dependent stress response among Archaea (Hartzell et al., 1999). In bacteria, this is a typical response to stress conditions. Interestingly, we observe extensive cell aggregates of both H. lacusprofundi and M. burtonii when cultures are incubated in the cold (Figure 4.1). This is of particular interest since the formation of a multicellular complex, comprised of archaeal, bacterial, or eukaryotic cells, constitutes one of the most fundamental aspects of developmental biology. Cell aggregation may facilitate exchange of nutrients, membrane components, and genetic material, thus enabling the organisms to cope with a stressful environment. 4.2.4.1 Biofilm studies Many microorganisms, including Archaea, are present in microbial mats found in hypersaline environments (Spear et al. 2003), anaerobic methane oxidation systems (Boetius et al. 2000; Orphan et al. 2001; Hallam et al. 2004) or acid mine drainage (Tyson et al. 2004). The latter two examples addressed interactions between members of the domains Archaea and Bacteria using environmental genomics. Archaea-containing biofilms are also important in industrial applications (Hulshoff Pol et al., 2004; Zhang et al., 2003), and in the hindguts of many terrestrial arthropods (Eckburg et al. 2003). However, knowledge about molecular details of biofilm formation in the Archaea is sparse. Means for surface attachment may involve unique flagella that have structural similarities to the type IV pili of bacteria (Thomas et al. 2001) as well as other proteins predicted to be involved in fibril formation and surface adherence (Kachlany et al. 2000). Various exopolysaccharides, key components of biofilm-matrices, are synthesized by halophilic and thermophilic Archaea (Rinker and Kelly, 2000). Mainly due to a lack of an appropriate model organism, many important aspects such as cell-to-cell communication and intracellular signaling pathways in archaeal biofilms have not been addressed so far. As these are fundamental to microbial life on Earth, it is likely that microbes on other worlds will also exploit identical or similar strategies for survival. Our recent experiments demonstrate enhanced biofilm formation for two psychrophiles, H. lacusprofundi and M. burtonii. Since biofilm formation by microbial cells is a genetically programmed process (Lazazzera 2005), microarrays are particularly promising tools to gain insights into the underlying mechanisms. Metabolic pathways of biofilms can also be explored based on proteomic approaches. A community based proteomics has been applied to investigate the microbial functions associated with acid mine drainage bacterial biofilm (Ram et al. 2005), and bacterioplankton in the estuarine water (Kan et al. 2005). Specific aims: (1) Address cold-adaptation and biofilm formation during growth at low temperatures by recording the transcriptome of H. lacusprofundi and M. burtonii using a time lapse. (2) The extracellular matrices will be purified and chemical composition determined. Experimental plans: We plan to use in situ synthesized oligonucleotide arrays for both H. lacusprofundi and M. burtonii, using ink-jet technology (Hughes et al. 2001). We previously used this technology successfully for transcriptome analysis of DMSO/TMAO respiration and response to UV irradiation in Halobacterium sp. NRC-1 (Müller and DasSarma, 2005; Confidential Page 36 2/12/2016 McCready et al., 2005). Oligomer (60-mer) probes will be designed for all ORFs utilizing the program OligoPicker (Wang and Seed, 2003). The 11,000-feature capability allows for design of multiple probes per ORF with similar mean Tm and narrow range (<3C) as well as ample negative and positive control spots to test hybridization conditions. A hallmark of in situ synthesized oligonucleotide arrays is the low occurrence of data outliers due to non-uniform spot morphology or background noise. Features replicated within a single array show low differences in absolute processed signal intensities and spot-to-spot variation for replicate experiments. Microarray experiments will be conducted in collaboration with J. Müller, Morgan State University. The genome sequence of M. burtonii is currently being constructed by DOE-JGI in collaboration with CoI Sowers (see http://genome.jgi-psf.org/) and closure is estimated within 6 months. A microarray will be constructed and parallel studies will be conducted with the methanogen to identify genes expressed in response to cold growth. Employing oligonucleotide microarrays, we expect to gain knowledge of the mechanisms of cold-adaptation in H. lacusprofundi and M. burtonii on several levels. (i) A principal analysis of the effect of cold temperature regimes on the transcriptome this organism will occur. Additional temperature-dependent regulatory networks and promoter analysis will be supported by comparative genomics. (ii) An insight into gene activity on a global scale will be obtained. Averaged signal intensities of probes are approximately proportional to the relative transcript level of the corresponding gene. For example, the genome of H. lacusprofundi contains ca. 10 csp genes, and we will be able to address hierarchy in importance of the CSPs. (iii) The comparison with existing microarray data on cold-adaptation in Halobacterium sp. NRC-1 will reveal what is unique to H. lacusprofundi in its response to cold temperatures. Is there a subset of genes that is present in both organisms but differentially expressed only in H. lacusprofundi? Or are there genes differentially expressed in H. lacusprofundi that are not present in Halobacterium sp. NRC-1? Similar comparative analysis may also be conducted for the psychrotolerant methanogen M. burtonii and the mesophilic methanogen, and M. acetivorans. A second avenue of studies will address biofilm formation in H. lacusprofundi and M. burtonii during growth at low temperatures. Since biofilm formation by microbial cells is genetically programmed, microarrays are particularly promising tools to gain insights into the underlying mechanisms. Thus far, no report on global gene expression changes associated with biofilm formation in Archaea has been published. We plan to record the transcriptome of H. lacusprofundi and M. burtonii during different times of biofilm formation. Incorporating several points is of particular importance since there is no clearly defined ‘endpoint’ in biofilm formation (Lazazzera 2005). The experimental approach will be similar to that previously established for various Bacteria (Beloin et al. 2004, Pysz et al. 2004, Resch et al. 2005). Cells grown in biofilms will be compared with planktonic cells from the same cultivation vessel as reference. We expect to identify the subset(s) of genes important for biofilm formation and maintenance, and gain an insight into the regulation of those genes. We are aware that cell populations in biofilms are rather heterogeneous, which may complicate data analysis. We plan to study the composition of extracellular matrices visible in biofilms of H. lacusprofundi and M. burtonii (and possibly other psychrophilic microbes), with the goal of identifying their chemical composition. Initial results indicate that a polysaccharide is present, at least for H. lacusprofundi, based on Congo Red staining (Allison and Sutherland, 1984). Similar Confidential Page 37 2/12/2016 studies will be conducted with M. burtonii to determine if analogous mechanisms may be used in both of these psychrophiles. For further analysis extracellular matrices will be purified by via tangential-flow filtration (10,000-molecular-weight cutoff) and precipitation and size-exclusion chromatography and subjected to enzymatic and mass spectrometry analysis (Maira-Litran et al. 2002; Vuong et al. 2004). 4.2.4.2 Gene transfer and complementation studies Different groups of microorganisms exhibit distinct types of cold temperature responses (Inouye and Phadtare, 2004). For example, E. coli responds to low temperatures by adjusting its membrane lipid composition and increasing the production of both CSP proteins and proteins that facilitate ribosomal assembly. In contrast, yeast responds with increases in the synthesis of trehalose and a heat shock protein. Genomic analysis of H. lacusprofundi and M. frigidum Ace-2 has identified stress proteins that are likely to be important for survival of these species in the cold, including a number of cspD homologs (our unpublished data). Our strategy is to identify genes that can promote cold growth and survival by gene transfer from the psychrophilic to the mesophilic species. For the halophile, we plan to use the model, Halobacterium sp. NRC-1, as host, while for the methanogen we will use M. acetivorans. Both mesophilic species have welldeveloped genetic systems in use in our laboratories as well as complete genome sequences, making them ideal for complementation analysis. Specific aims: (1) To examine what protection cspD genes provide to cells exposed to low temperatures. (2) For complementation analysis, use additional genes identified through bioinformatic and microarray studies, e.g. anti-freeze protein genes and lipid biosynthetic genes, which may confer the ability for cold growth. Experimental plans: We plan to overexpress a selection of cold-active genes identified in the psychrophiles in the mesophilic model counterparts to address and confirm the involvement of those genes in cold adaptation. Initially, we will overexpress the CSP genes of the psychrophiles in both organisms. A previously constructed and tested overexpression vector, pKJ408 (Jung and Spudich, 1998), will be used for the halophile system in the DasSarma lab. In pKJ408, the cloned gene will be under control of the ferredoxin promoter from Halobacterium sp. NRC-1 to enable moderate overexpression. In addition, pKJ408 harbors a mevinolin resistence gene, mevR, for selection. PCR primers will be designed for amplification of the respective gene plus introduction of appropriate restriction enzyme sites (NdeI and XbaI). PCR-amplified genes will be restriction digested and ligated into restriction-digested pKJ408. The resulting vector will be amplified in E. coli and subsequently transformed into H. lacusprofundi and Halobacterium sp. NRC-1 essentially as described (DasSarma et al. 1995). Successful overexpression will be tested for by semi-quantitative Reverse-Transcriptase-PCR and by standard SDS-PAGE protein analysis. If required, a new vector will be constructed in which the ferredoxin promoter from Halobacterium sp. NRC-1 will be substituted with that from H. lacusprofundi. The physiological response of either organism after successful CSP expression will be tested by standard growth tests. Subsequently, we intend to express additional genes after they have been implicated to be involved in cold adaptation. The effects of CSP in the mesophile will be tested by expressing the gene in a plasmid-based expression system developed by CoI Sowers for Methanosarcina acetivorans. The vector pSM1, Confidential Page 38 2/12/2016 a derivative of pWM315 (Metcalf et al, 1997), contains a sequence of the native plasmid from M. acetivorans, p2CA, to promote autonomous replication in M. thermophila -pir dependent R6K origin of replication for amplification in E. coli. The parent plasmid also contains the puromycin resistance gene (pac), which confers puromycin resistance for selection in M. thermophila and the gene for -lactamase for ampicillin resistance in E. coli. The CSP will be cloned downstream of the promoter for the gene encoding CO dehydrogenase in M. acetivorans, which is expressed in cells grown with acetate. If flanking restriction sites are not compatible with the multiple cloning site of the expression vector, compatible sites will be inserted by PCR-mediated mutagenesis of one or both flanking regions. The construct will be transformed into E. coli and the cloned gene will be confirmed by restriction analysis and sequencing. The expression vector will then be transformed into M. acetivorans and confirmed transformants will be tested for extended tolerance to low temperatures. This system has already been shown to effectively express other archaeal proteins (Sowers, manuscript in preparation). 4.2.4.3 Ecological and proteomics studies A substantial fraction of Earth’s biosphere undergoes seasonal freeze-thaw cycles. Among the diversity of cold adapted bacteria found in these environments, the genome-based study of Colwellia psychrerythraea (Methe et al. 2005) recently showed that cells undergo a collection of synergistic changes when growing in cold condition, which include DNA and amino acid composition, cell membrane fluidity, uptake and synthesis of compounds conferring cryotolerance, and cold-adapted enzymes for carbon uptake. A significant priority will be increasing our understanding cold-adaptation of microorganisms. For this goal, we plan to expand our collection of cold-adapted species and consortia with samples from the study sites of Co-I Robb, whose Microbial Observatories funded project in the Uzon Caldera, Kamchatka Peninsula has permafrost locales on the rim of the caldera. Also, samples will be collected in the Svalbard Mars analog study site via collaborators Fogel and Steele of the CIW NAI, and additional polar sites, in the Antarctic Vestfold Hills and Ace Lake (via our collaborator R. Cavicchioli at the University of New South Wales). Additional studies with collaborator Onstott will focus on a deep permafrost site in Nunevat Territory, Canada as a Mars analogue site where we will be able to obtain permafrost core and saline brine to a depth of 0.5 to 1.1 kilometers. These samples will be enriched using the microbiology procedures we are developing for the model organisms, including growth under progressively declining temperature regimes to determine the Tmin for growth (Sowers et al, in preparation). We will compare and contrast new isolates with the physiological types we are studying, and the existing culture collection at COMB. CoI Chen has compiled a large collection of bacterial strains (incluing Synechococcus spp. CBO101 and CBO205) from the Chesapeake Bay, isolated during the winter season at 0 oC, as well as during warmer seasons (Kan et al. in press). Most bacterial strains isolated in winter are distinct phylogenetically from summer isolates, and many are closely affiliated with isolates from colder and permanently cold environments including polar seas, Arctic sea ice, Antarctica, Baltic Sea etc. Since the Chesapeake Bay isolates are adapted to the winter surface water which may sometimes be frozen, their cold adaptation mechanism is of significant interest. Comparison between these isolates from a temperate climate with those from Kamchatka Peninsula, Svalbard, and from Antarctica will provide information of the similarities and differences in cold-adapted Confidential Page 39 2/12/2016 microorganisms. A combination of culturing and comparative proteomics will be used to explore the major proteins responsible for psychrophilic activities. Specific aims: (1) Environmental isolates will be tested for growth at the lowest possible temperatures. (2) Winter isolates from temperate climate will be compared to those from permanently cold Arctic and Antarctic environments. (3) Physiological responses to low temperature at the protein level will be investigated using proteomics. Experimental plans: All environmental samples will be stored and transported frozen to CoI laboratories at COMB (Chen, Robb, Sowers, and DasSarma) under appropriate conditions. Bacterial strains (over 200 strains) previously isolated from winter and summer expeditions in the Chesapeake Bay are currently preserved at –80oC. These strains will be cultivated at different temperatures to test for the lowest possible temperature for growth, as described in sections 4.2.1-3. Meanwhile, bacterial strains from indigenous Arctic and Antarctic environments will also be tested to compare the psychrophilic activities between temperate and polar environments. A model enzyme, glutamate dehydrogenase, will be assayed at a wide range of temperatures as a reporter of the temperature optimum of the proteome. This will greatly expand our availability of cold-adapted species of microorganisms for understanding mechanisms of cold adaptation. Comparative proteomics will be exploited to understand the key proteins corresponding to the psychrophilic activities. Proteomics and comparative proteomics have been applied to explore expressed proteins from the whole organism (Blackstock and Weir 1999, Lopez 1999) or whole community (Kan et al. 2005, Ram et al. 2005). Unique proteins produced in response to a specific environmental change can be identified by comparing the protein expression patterns between control and treatment. CoI Chen and DasSarma routinely use 2-D gel based proteomics in our labs at COMB and additional expertise is available through collaborator R. Cavicchioli’s lab at USNW and the UMB Proteomics Core facility. Briefly, cultures grown at different temperatures will be harvested by centrifugation. Protein extraction, 2-D gel electrophoresis, image analysis, protein spot excision and characterization will follow the protocols described by Kan et al. (2005). Application of proteomics permits study of the physiological and biochemical reaction of bacteria under the tested environmental conditions. Genomes of several bacteria included in this proposal have been sequenced, making comparative proteomics more feasible. Initial efforts will be devoted to setting up the culture systems for psychrophilic microbes, since they grow slowly. Subsequently, we plan to conduct the environmental proteomics studies in order to understand mechanisms of cold adaptation. 4.3 Radiation tolerance, species survival and meteoric transfer Mars and Europa, the two likeliest candidates for extraterrestrial life in the Solar System, possess low-pressure atmospheres, which offer little protection against desiccation and high-energy irradiation. Moreover, meteoritic transfer of material between Mars, Earth and the Jovian system has taken place over the lifetime of the Solar System (McGenity et al, 2000; Mileikowsky et al, 2000; Melosh, 2003). There have been suggestions that life could have originated on the more benign environment of a young wet Mars, and then contaminated a young Earth (interplanetary panspermia). Indeed, interstellar transport may be possible in young star clusters (see Section 4.3.2). Confidential Page 40 2/12/2016 At the present time, the Sun is a relatively inactive star, but exoplanets in other systems are likely to be subject to a more potent flux of high-energy radiation, while the Sun itself was more active at a younger age. The diversity of organisms that can survive low-pressure conditions is crucial for addressing the potential for life on Mars-like planets with low-density atmospheres, life on M dwarf exoplanets (see Section 3.4), the viability of interplanetary seeding and the potential for established ecosystems to recover from short-term exposure to high-energy radiation. 4.3.1 Radiation sensitivity of extremophiles Radiation-tolerance of microorganisms is likely to serve as a significant advantage for survival on planets subjected to high radiation intensities and is also relevant from the standpoint of planetary protection, especially for Mars (e.g., Cockell et al, 2000). It also factors into the feasibility of panspermia mechanisms. Several microorganisms are known to be radiation resistant, a trait usually correlated with desiccation resistance. Examples include both thermophilic and halophilic Archaea as well as photosynthetic cyanobacteria. Depending on the species and strain, resistance may be observed to UV light (including the most damaging UV-C) as well as high-energy radiation (including gamma radiation and beta-particles). For some species, LD37 levels may be greater than 200 J/m2 for UV radiation and 10K Gy for high-energy radiation. While DNA repair processes of common laboratory species, such as E. coli and yeast, are well studied, the repair mechanisms of extremophilic species are largely unknown. We (CoIs DasSarma, Robb, Chen and collaborators) are interested in the mechanistic basis of radiation resistance in several extremophiles, including the halophilic species, Halobacterium NRC-1, and the thermophile, Pyrococcus furiosus. These organisms display somewhat different genomic features and resistance mechanisms. For the halophile, genomic studies have shown nucleotide excision repair systems related to both bacteria (e.g. UvrACBD exonuclease) as well as the eukaryotic type (Rad2, 3, 25, etc.) (DasSarma et al. 2001). A combination of whole genome microarray studies (McCready et al., 2005) and gene knockouts shows that both systems are in use. Moreover, DNA repair likely involves induction of homologous recombination systems, which may allow repair of double stranded breaks and DNA replication arrest points. The repair mechanisms of P. furiosus are interesting since this archaeon grows optimally at 100 o C and is unlikely to encounter ionizing or UV radiation during normal growth (DiRuggiero et al, 1998; Saffary et al. 2002). However, constant growth at or above 100 oC induces both doubleand single-strand breaks into DNA in vivo, and we recently found that heat shock per se induces DNA repair capacity, and several important DNA repair enzymes, such as the recombinase radA. The importance of radA induction has also been observed in Halobacterium sp. NRC-1 after UV irradiation (McCready et al. 2005). Cyanobacteria must cope with the negative effects of ultraviolet stress caused by their obligatory light requirement for photosynthesis. They may have been present 3.5 billion years ago (Schopf 1993), suggesting that they were able to live in an O2 deficient but UV intense environment. Some cyanobacteria contain effective radiation repair mechanisms such as synthesis of UVabsorbing scytonemin, secretion of copious extracellular mucilage, phototactic motility and adherence to substrates. Photoautotrophic cyanobacteria can serve as a model organism for studying the tolerance of radiation (Hader et al. 1998, Ehling-Schulz and Scherer 1999). Repair mechanisms can take the form of DNA repair (Blakefield and Harris 1994) or de novo synthesis of D1 and D2 proteins, the key proteins for repair of UV-B-induced damage in photosystem II Confidential Page 41 2/12/2016 (PSII) (Vass et al. 1999, 2000). A freshwater cyanobacterium, Synechococcus sp. is able to resist UV-B radiation by switching to two other forms of D1 proteins (Campbell et al. 1998). Many marine Synechococcus strains with different pigment types have been isolated and maintained in the Chen lab; selected Synechococcus strains will be used for radiation tolerance experiments. Specific aims: (1) Select mutants with increased high energy-radiation tolerance level for Halobacterium sp. NRC-1, Pyrococcus furiosus, and marine Synechococcus. (2) Characterize their physiological and molecular responses to the UV radiation. Experimental plans: To better understand the basis of radiation tolerance, we will select derivative strains with increased high energy radiation resistance. Preliminary studies conducted using the LINAC facilities available at the Idaho Accelerator Center (IAC) with the halophile, Halobacterium sp. NRC-1, show that mutants of that species are more highly resistant than Deinococcus radiodurans (DeVeaux et al. 2005). Linda DeVeaux at the IAC will serve as our collaborator for these experiments. Stationary phase cultures of Halobacterium sp. NRC-1, P. furiosus, and marine Synechococcus will be exposed to increasing doses of 18-20 MeV electrons from a pulsed LINAC. Survivors will be regrown to stationary phase and subsequently irradiated through multiple cycles. Dose-dependent survival for isolates will determined by comparing colony-forming units of irradiated cultures to unexposed cultures. Similarly, we have also recently determined that P. furiosus has an increased survival rate at very high fluxes of radiation from a Co60 source after sublethal heat shock; we will repeat these experiments with pulsed 18-20MeV electrons, comparing survival in cultures under optimal growth conditions (100oC) and heat shock (60 minutes at 105oC). Viability after irradiation will be determined by end point dilution assay (DiRuggiero et al, 1998). The objective will be to determine the role of the heat shock inducible DNA repair systems of hyperthermophiles under high energy electron bombardment. Analogous experiments will be conducted with selected marine Synechococcus isolates. Subsequently, we will examine chromosome fragmentation and reassembly using pulsed field gel electrophoresis, as in previous studies with the Co60 source. An additional goal will be to understand the physiological responses of the radiation resistant microorganisms to UV irradiation. We plan to utilize whole genome microarrays, as established for Halobacterium sp. NRC-1 (Section 4.2.4.1) as well as CPD assays. A variety of tools, such as antisera for CPD assays are available through our collaborator, Shirley McCready, at Oxford Brookes University. Our goal is to determine the critical response of a diversity of UV-tolerant cells to DNA damage via UV radiation. 4.3.2 Panspermia in young systems Interstellar transport of material is extremely unlikely among field stars (Melosh, 2003), but transfer rates among young stars in open clusters (the birth place of most stars) is orders-ofmagnitude higher (Adams & Spergel 2005). If transfer is possible (and likely), then the implications for our understanding of the spread and origin of life is profound: life could originate in only a handful of systems, yet the existence of life could be ubiquitous. Are young solar systems fertile environments for the propagation and growth of life? We will address this question by carrying out a series of linked investigations spanning from astronomy to biology: Confidential Page 42 2/12/2016 How long can life survive in an open cluster? Basing our analysis on various sites of ongoing star-formation (e.g., Orion, Taurus-Auriga), the Princeton group will model the ultraviolet and cosmic ray environments in open clusters. These variables will serve as input for the experimental work outlined in the following section, where extremophiles will be exposed to high radiation environments to determine their survivability. Do pre-biotic molecules survive the planet formation process? There has been significant interest in the detection of pre-biotic molecules in molecular clouds (see Sanford, 2005). Will complex molecules survive the proto-stellar environment? Where in the early solar system can they survive? What is the rate of cometary delivery of these molecules? What is the transfer rate between planets during the early and late bombardment phases? If life originated on Mars, what planets and moons could be seeded with Martian life? What if life originated on Europa? What are the transfer rates between environments and other solar systems in an open cluster? How does the dynamic focusing effects of Jupiter alter these rates? We will extend the numerical analysis of Adams & Spergel (2005) to these systems. When do planets become habitable and what is the range of habitable environments? In our early solar system, Mars was likely a far more hospitable environment: warmer and with higher atmospheric pressure. On the other hand, most forms of life could not survive in the early Earth. We will synthesize our panspermia studies with results from relevant investigations by NAI teams (notably Woods Hole, Penn State, Arizona and Colorado) to determine the optimal epochs and locations for cross-fertilization.. The standard view of the origin of life suggests that life originated on the early Earth very shortly after it became sufficiently cool. If so, panspermia in young clusters could lead to life forming spontaneously and independently on each habitable planet in a solar system. If the transfer rate of life within the solar system is high enough, our solar system studies may find life forms on other bodies that are closely related to terrestrial life, as discussed further in Section 4.4.2. 4.4 Test cases in the Solar System 4.4.1 Species survival on Mars The survival of endemic and/or transplanted microbial life in the near-surface environment of Mars must involve the interaction between microbial community and the sources of carbon, nutrients, and energy in the Martian regolith and fractured bedrock. The COMB team will test adaptation of terrestrial extermophiles to conditions that simulate various aspects of the Martian regolith and bedrock. In previous sections of this proposal, we discussed exploring the cold temperature limits for a variety of psychrophiles. These experiments will also involve control of headspace gas compositions, in some cases to reflect interactions between the Mars atmosphere and microenvironments in the Mars subsurface. In this section, we discuss appropriate liquid media and solid phases to use for testing species survival and adaptation to Mars-like conditions. A widely used simulant material for the Mars surface environment is the JSC Mars-1, which consists of poorly crystalline volcanic ash of basaltic composition from the Pu’u Nene cinder cone, Hawai’i (Allen et al., 1997). This ash source was chosen as a Mars regolith simulant because of the similarity in the visible and near infrared the reflectance spectra between the simulant and high emissivity (i.e., “bright”) regions of the Mars surface. However, since the Confidential Page 43 2/12/2016 Martian regolith has been exposed to weathering processes for potentially hundreds of millions of years or longer, such chemical alteration would push this regolith to a composition that is chemically and mineralogically more diverse than unaltered basalt. A broad cross-section of the Mars research community has recognized that a single simulant is insufficient to characterize the wide range of inhabitable chemical environments that may have existed and/or that may yet persist on Mars. In microbiology and astrobiology, a liquid medium derived from “rinsing” or overnight soaking of the JSC Mars-1 simulant in DI water has been used. Modeled Mars brine chemistries could also be used for enrichment cultures. Several magma derived minerals and their weathering products have been detected or are inferred to exist on the Martian surface (e.g., zeolites, smectites), though we are not aware of their use in psychrophile culturing work. Another set of studies uses Mars Analog Environments on Earth as a starting point for assessing natural microbial communities and preparing enrichment cultures, sometimes in media based on the particular analogue environment sampled. Three such Mars analogue sites will provide our group with field enrichment cultures for various psychrophile metabolisms. We plan to utilize samples from cold environments investigated by our CIW collaborators, Andrew Steele and Marlyn Fogel, through the NAI-funded Arctic Mars Analogue Svalbard Expedition (AMASE). These environments possess a range of basaltic lithologies and include the outflows of geothermal fluids that rapidly chill and provide disequilibrium chemistries in support many different metabolisms. This work will be conducted through our collaboration with Steele and Fogel, who have already provided F. Chen at COMB with sample material for enriching psycrhophilic endolithic cyanobacteria. We will obtain further samples for halophile, methanogen, and cyanobacteria cultures from Antarctica, via our collaboration with R. Cavicchioloi, UNSW. We will also collaborate with TC Onstott, Princeton, to obtain samples from the deep permafrost Mars Analogue site being developed in the Nunevat Territory, Canada, by the Indiana-Princeton-Tennessee Astrobiology Institute (IPTAI). This site will be cored to a depth of 0.5 to 1.1km to recover deep permafrost and cold brine samples. In addition to chemical composition, the availability of water will have a mitigating influence on the presence of viable life. A prior report tested the ability of three mesophilic (25-55 oC), hydrogen utilizing methanogens to grow in a buffer containing a Mars soil simulant comprised of altered volcanic ash containing different water concentrations (Kral et al, 2004). Results showed that these species produced methane in the simulant under a carbon dioxide-hydrogen (95:5) headspace. All three also generated methane in simulant without standing liquid, although the effect varied with species. This study measured methanogenesis but did not confirm growth of the methanogens. Martian soil experiment Specific aims: (1) To measure metabolic activities and growth of representative archaeal strains under Mars-like conditions. The main emphasis will be on ionic composition of the medium, mineralogic and chemical composition of the regolith simulant, and water content of the regolith itself. (2) Establish enrichments for novel Bacteria and Archaea that thrive at high osmotic strength of the medium and low to circumneutral pH. Confidential Page 44 2/12/2016 Experimental plans: Experiments will include tests for growth and metabolic activities of test microorganisms in simulants from different locations and using a range of water content. We will employ both traditional soil and soil water simulants (e.g., JSC Mars-1 and DI rinsate) and simulants that explore other proposed habitable environments in the Mars subsurface (NaCl brines and CaCl2 brines reacted with JSC Mars-1; ferric sulfate-rich brines at low pH; media at low to circumneutral pH in contact with individual constituent minerals of basalts as well as smectites and zeolites. In addition, Mars’ surface area contains high levels of iron oxides, which could serve as an electron acceptor for dissimilatory iron reducing prokaryotes. Therefore, we will also establish multiple enrichments to test for psychrophilic methanogens that can grow in water containing only selected simulants and hydrogen/carbon dioxide and dissimilatory iron reducting halophilic Archaea. Sediments from Arctic, Antarctic and permanently cold marine sediments will be used as inoculum for cold adapted methanogens and cold halophilic sources (e.g., Deep Lake) will be used as inoculum for halophiles. Methanogens will be monitored by measuring methane production (metabolic activity) and by competetive PCR of 16S rRNA (growth). Halophile growth will be monitored by plating. Once stable enrichments are obtained, isolates will be obtained by standard dilution methods carried our either under oxic or anoxic conditions. Isolates will be characterized by classical- and molecular microbiological methods. The inocula for these enrichment cultures will derive from sampling in association with our collaborators: Svalbard samples with Andrew Steele and Marilyn Fogel and the AMASE team, Antarctic samples with R. Cavicchioli, and Nunnevat Territory, Canada, deep permafrost and brines with TC Onstott and IPTAI. 4.4.2 Terrestrial contamination of the Solar System Just as Martian material reaches Earth, large impacts distribute terrestrial material through the Solar System. Nearly 5% of rocks ejected from Earth eventually reach the surface of Mars and ~1% reach the Jovian system (Melosh, 2003). These ejecta may transport terrestrial microbes, and therefore have the potential to contaminate (and even seed) Mars or Europa, the two prime astrobiological targets within the Solar System. Previous work has characterized ejecta number and mass, and survival of a limited number of microbial organisms in transit; the experiments outlined in Section 4.3.1 will expand our knowledge on the latter question. No study, however, has examined what fraction of Earth-derived meteoroids impact the Martian and Europan surfaces in a way that permits microbial survival. We consider that issue here. Mars Impact survival: The low-density Martian atmosphere acts as a filter to meteoroids: some ablate completely; large meteoroids airburst; and still larger ones experience little deceleration and strike the surface at hypervelocities (Chyba, 1993; Chyba et al., 1993; Mileikowsky, 2000; Melosh, 2003). It is likely that this substantially reduces the total mass of viable microorganisms that reach the Martian surface—though that number will certainly not be reduced to zero. We propose to carry out a quantitative analysis, with the ultimate goal of establishing a lower limit for background levels of natural biological contamination on the surface of Mars. Our work will build upon numerical codes for the Earth’s atmosphere used to examine incoming bolides of varying densities and sizes (Chyba, 1993; Chyba et al., 1993). Recent data from the Mars Global Surveyor, Climate Orbiter, and Exploration Rover missions will be used to provide input data for the atmospheric profiles. A range of bolide atmospheric entry incident angles will Confidential Page 45 2/12/2016 also be examined and the resulting aerobraking and ablation will be incorporated to estimate the temperature evolution during entry through the Martian atmosphere. Thus, for a given mass, density, entry angle, entry velocity, and microbial contamination level, our code will be able to predict the fraction of viable microbes delivered to the Martian surface. This work will be supervised by Chyba and pursued by his graduate student, Mr. Kevin Hand. This analysis will not only enhance our understanding of microbial transfer between planets but will also prove of interest for analyses of what level of “hitchhiking” microbes are permissible for spacecraft traveling from Earth to Mars. Europa Europa is the premier place in our solar system to search for a separate origin of life. The lack of atmosphere, added distance, and radiation of the Jovian system may well protect the sub-surface liquid water environment from contamination by inhabited Earth-rocks. As a result, if we want to learn the most about the diversity of life, Europa is the place to explore. Here we propose to calculate the background biological contamination level experienced by Europa over its history. Several stages leading to contamination must be considered: (1) delivery of a contaminated Earth rock to the Jovian system, (2) delivery to the surface of Europa, (3) retention of material vs. loss during impact (Pierazzo and Chyba, 2000), (4) delivery of material directly to the putative subsurface ocean via complete penetration of the ice shell (Turtle & Pierazzo, 2001), and (5) survival time of viable biological material on or near the surface as compared to the shell resurfacing timescale. For some stages, useful work has been published. However, many need more complete analysis and a synthesis of all stages has never been attempted. We (CoI Chyba and Hand) will build on previous simulations of meteoritic transfer to Europa to provide a quantitative analysis of natural background contamination for both the surface and sub-surface of Europa. 4.5 Summary The principal tasks described in this section are as follows: We will use comparative experiments to study the adaptation to cold environments of representative extremophile species. We will investigate mechanisms for cold adaptation using microarrays, gene transfer and proteomics, and search for cross-species similarities. We will examine the radiation sensitivity of key extremophiles, and determine their survival potential in the high-UV radiation environments on Mars-like planets and in young star clusters. We will examine the growth potential of key terrestrial extremophiles in simulated Martian environments. We will investigate the potential for terrestrial contamination of Mars and Europa. Confidential Page 46 2/12/2016 5. How will we recognize if life is present? 5.1 Introduction Goal #7 of the Astrobiology Roadmap centers on identifying and recognizing signatures of life. This is also a key aspect of NASA’s Vision for Space Exploration. Within the Solar System, the capacity to sense life remotely would allow surveys of local environments, such as the surface of Mars and the surfaces of astrobiologically interesting Solar System moons, even with ground based instrumentation (Sparks, Hough & Bergeron, 2005). The next logical step, given such a capability, is to include appropriate instrumentation as a payload element in spacecraft Solar System exploration, such as Mars orbiters and the Jupiter Icy Moons missions. In this way, vast areas of planet and moon surfaces can be surveyed for direct detection of life, and it is straightforward to imagine such a procedure being a fundamental part of site selection for in situ experiments made with landers and field instrumentation. Beyond the Solar System, remote sensing is an absolute necessity and NASA is developing TPF to seek direct images and spectra of Earth-like planets. Currently our best remote sensing “biomarkers” are based on the ability of life to modify atmospheric composition (Woolf & Angel 1998). NAI researchers (notably at NASA Ames and CIW) have devoted considerable effort to exploring potential spectroscopic biosignatures, such as O2 and the red edge due to chlorophyll absorption (Seager et al, 2005). These features can be examined most easily in a planetary context through observations of the Earth itself, and we describe a program of Earthshine observations later in this section. However, the interpretation of spectroscopic biosignatures, and consequent inferences on the presence of life, are substantially model dependent. It is important that we begin to build a more sophisticated understanding of the likely many ambiguities that can arise. If an unambiguous biomarker exists, it is vital that we know it! Circular polarization is such a candidate. 5.2 Validating circular polarization as a biosignature in terrestrial environments Circular polarization has been discussed in the astronomical literature for over 30 years as a potential biosignature (Pospergelis, 1969; Wolstencroft, 1974). Over the last 18 months, we (CoIs Sparks, DasSarma and Chen, and consultant Germer) have been investigating circular polarization’s potential as a biosignature. Our initial laboratory experiments suggest that circular polarization of light scattering from biological material can be orders of magnitude larger than from abiotic material; this is consistent with prior work. We have begun astronomical application of this technique, with imaging polarization of the Martian surface during the favorable 2003 opposition (Sparks et al, 2005). But the most effective means of proving circular polarization as a biosignature is through measurements of targets that unequivocally include living material; that is, measurements of environments here on Earth, in tandem with control abiotic samples. We will verify the effectiveness of circular polarization as a biosignature through 1. Measurements of key natural substances in the laboratory 2. Measurements of terrestrial scenes in the field. As part of this NAI proposal, we will construct a circular polarimeter instrument designed specifically for in situ terrestrial measurements. If we can demonstrate that circular polarization Confidential Page 47 2/12/2016 works as a life detector on Earth, then application to future space missions, both interplanetary probes and exoplanet observatories, is potentially straightforward yet incredibly important. 5.2.1 Circular polarization and life Chirality of living matter Life is predominantly homochiral. Almost all organisms use only left-handed or L-amino acids and right-handed or D-sugars. This is likely a necessity for self-replication and hence will characterize all biochemical life including currently unknown forms. Intriguingly, analysis of the Murchison meteorite has shown L-excesses of 2-9% for a number of -methyl amino acids (Cronin & Pizzarello 1997), with slightly smaller excesses found in the Murray meteorite (Pizzarello & Cronin 2000). The origin of this phenomenon is not known; it could be an amplified relic of an ancient excess, such as might be found in the interstellar medium or prebiotic matter delivered to Earth, or it may arise from a surface interaction that favors one handedness over the other. Nevertheless, it is a “universal” quality of terrestrial life, and chirality studies have been proposed as a robust means of searching for life that may indeed differ from terrestrial life, except in this regard. Living matter can induce circular polarization Organic material can be optically active, arising from differential absorption or scattering of left and right circularly polarized light by its component chiral molecules (Pospergelis 1969, Wolstencroft 1974, Winebrenner & Ashner 2000, Wolstencroft et al. 2002). Circular dichroism spectroscopy is a standard analysis technique for studying protein structure (Purdie & Brittain 1993, Kelly & Price 2000). Chlorophyll, for example, induces 0.1- 1% circular dichroism in its absorbance bands (Houssier & Sauer 1970). Circular Intensity Differential Scattering (CIDS) considers the circular polarization induced by scattering from chiral samples and has been studied by Bustamante et al. (1985). Circular polarization can also be caused by optical interaction associated with the birefringence of macroscopic biopolymer membranes of an organic structure, while light emitted through fluorescent processes, commonly associated with molecules of biological significance, may also exhibit circular polarization. Microbes typically produce fractional polarizations ~10-2 to 10-3 (Salzman & Gregg 1984). Here, we are interested in the possibility that interaction between light and living organisms can produce a potentially detectable signal ~0.1% or more, in the form of induced circular polarization. Abiotic contributions to circular polarization arise from atmospheric and aerosol scattering and from mineralogical scattering processes. While some minerals and crystals are certainly optically active, integrating over a naturally produced sample is expected to give equal fractions of enantiomorphs that average to zero in their optical activity. Multiple reflection and phase effects can also introduce circular polarization; however, those would produce a smooth distribution recognizably related to the geometry of the scattering (Bandermann et al. 1972). Empirically, Solar System measurements of circular polarization consistently find very low fractional polarization levels, ~10-4 to 10 –5 (Kemp et al. 1971, Swedlund et al. 1973, Meierhenrich et al. 2002), some two or three orders of magnitude smaller than likely organic effects. Even if local enantiomeric excesses of optically active minerals were to exist, it is expected that they would be distinguishable from circular polarization having an organic origin by their spectral properties (Pospergelis 1969, Kelly & Price 2000). Fig. 1 shows the absorption and circular dichroism Confidential Page 48 2/12/2016 spectra of the chromophores of two primary microbial targets. Not only is there a substantial circular dichroism signal but also there is a distinctive sign-flip through the absorbance bands. It should be emphasized that biological signals are potentially two to three orders of magnitude higher than abiotic signals and that polarimetry is capable of very high sensitivity and accuracy. Fractional polarizations of ~10-6 are already achieved in current astronomical polarimetry thus circular polarization spectroscopy offers a high potential for producing an unambiguous biomarker. Figure 5.1. Left: Absorption spectrum of chlorophyll (red, solid) and circular dichroism spectrum (blue, dashed) adapted from Houssier & Sauer (1970). Right: Absorption spectrum (red, solid) and circular dichroism spectrum (blue, dashed) of bacteriorhodopsin (“purple membrane”), adapted from Becher & Cassim (1977). 5.2.2 Astrobiologically Relevant Samples Light Harvesting: vitally important, maximally observable An especially promising type of interaction between light and living organisms is photosynthesis (used broadly here to represent biological systems which “harvest” light). We begin our study by determining the circular polarization properties of important photosynthetic microbes. In photosynthesis, the organism extracts energy by triggering an electronic excitation of the photosynthetic pigment in diffuse absorbance bands. Circular dichroism and related phenomena can be induced from the intrinsic chirality of the molecules, from excitonic coupling between chromophores in molecular complexes and from macroscopic organization of the system (Garab 1996). From an astronomical perspective, photosynthetic activity is extremely attractive, since it is largely a surface phenomenon, which is required for external optical observation. Apart from the possibility of photosynthesis at ocean-floor hydrothermal vents, it requires interaction with light of the host star (including the Sun!), hence is tuned to the wavelength of maximum flux of the star and is tuned to the wavelength of maximum transmission of the planetary atmosphere (Wolstencroft & Raven 2002). These qualities combine to maximize the chances of detection through remote sensing. Photosynthesis is not limited to vegetation, but also occurs in primitive microbial life. We have therefore selected two of the most astrobiologically relevant microbial species for the first phase of analysis: Confidential Page 49 2/12/2016 Cyanobacteria are photosynthetic prokaryotes possessing chlorophyll a and their unique pigment phycocyanin. They have an extensive fossil record that may date back 3.5 billion years (Whitton and Potts 2000). With the early development of a photosynthetic capability, they are widely thought to have been the primary agent responsible for the rise of oxygen in the primitive atmosphere on Earth. Cyanobacteria are abundant and ubiquitous in marine and freshwater systems; are found in cold and hot environments; tolerate conditions of high desiccation; and can produce biochemical products that provide protection from UV damage. High diversity and great adaptability have made cyanobacteria extremely competitive on the planet Earth. These species have been studied extensively as part of exobiological research on the limits of life in the Solar system (Wynn-Williams 2000) and are now being studied as an analogue for past (or perhaps even present) life on Mars. Haloarchaea are salt-loving extremophiles, robust against UV radiation and tolerant of desiccation. They use a light harvesting system that contains the retinal-based chemical pigment bacteriorhodopsin in their metabolism. The tolerance shown by haloarchaea for harsh cold, dry, salty conditions make this species a candidate for life on Mars, should such exist at the present time (Landis 2001). Another example of the retinal-based rhodopsin system, proteorhodopsin, has been found in light-harvesting plankton (Beja et al. 2001). Figure 5.2 UV-VIS absorption spectra of chlorophyll (green) and retinal (purple)-based pigments. The complementarity of the spectra indicates that these pigments may have co-evolved (DasSarma, 2004; Wickramasinghe, 1976). The orange spectrum is for carotenoids. All known light harvesting systems require one of either chlorophyll or retinal-based pigments. A hypothesis proposed is that these two systems co-evolved, suggested by the complementarity of their spectra (see Figure 5.2). With their profound importance in astrobiology and potentially high observability, these organisms represent excellent targets for an initial study. We will use existing laboratory measurements where possible, and make new measurements where needed, to fully characterize the optical characteristics of light scattered by such organisms. Sample preparation and measurement strategies Our team has in-depth expertise on all aspects of the primitive photosynthetic haloarchaea and cyanobacterium organisms and permanent laboratory facilities dedicated to the culturing and Confidential Page 50 2/12/2016 study of the two proposed primary astrobiological targets, halobacteria (DasSarma) and cyanobacteria (Chen). For new measurements, our strategy will be to culture the microbial samples, which will be transported to NIST for polarization study. 5.2.3 Background and preparatory work Pilot studies at NIST (a) (c) (b) Figure 5.3. Pilot study from NIST showing circular polarization from 300 nm to 900 nm of reflected light from (a) halobacteria (b) a maple leaf (c) a control white mineral. Dark points use a blue sensitive PMT and lighter points, a red-sensitive PMT. The extra signal in the red for (a) may arise from fluorescence. The control sample upper limit is more than an order of magnitude lower than the detections. We have explored the measurement of circular polarization in the laboratory at NIST, experimenting with leaves and haloarchaea. We used a high precision photoelastic modulator (PEM), coupled with a monochromator and a photomultipler tube (PMT) detector, to measure the circular polarization of reflected light; some example results are shown in Figure 5.3. In a few sets of measurements, we found that blue and red sensitive PMTs gave inconsistent results (e.g., Figure 5.3a). This is probably due to organic fluorescence, but emphasizes some of the pitfalls of this type of measurement. In all cases, when we measured a control sample, we obtained null polarization at a level of 0.001%, consistent with expectations. In contrast, the biological samples gave polarizations a few tenths percent, at least an order of magnitude difference. These preliminary results underline the value of pursuing these investigations. Astronomical observations of Mars We have started to extend these measurements to bodies in the Solar System: we obtained high quality imaging circular polarimetry of the Martian surface during the extremely favorable opposition of 2003, (Sparks et al. 2005). We used the European Southern Observatory Very Large Telescope (VLT), one of the world’s forefront telescopes, in an imaging polarization mode that achieved polarization noise levels slightly below 0.1% and spatial resolution 210 km, but did not find any regions of circular polarization (Figure 5.4). This represents the best achievable results for current imaging astronomical instrumentation, and demonstrates the feasibility of the Confidential Page 51 2/12/2016 technique. Our observations covered only a small fraction of parameter space, so although we obtained a null result locally, we could not exclude the presence of optical activity at other wavelengths, in other locations or at higher spatial resolution, nor life in the wider sense. Figure 5.4 Observations of Mars. True color representation of VLT Mars observations (upper left) on cylindrical projection, with (lower left) outline of their footprint overlaid on USGS/NASA processed Viking image data. On the right, images of circular polarization degree (upper), displayed approximately 0.2% and absolute value of polarization degree (lower), 0 to 0.2%. The low level structure demonstrates a noise floor arising from internal interference within the CCD 5.2.5 Constructing a prototype field polarimeter We propose to construct a transportable prototype circular polarization instrument to make in situ measurements in a variety of terrestrial environments. The requirements to be determined for instrumentation will cover the spectral resolution and precision of polarization measurement needed in conjunction with the timescale on which measurements will be needed, and will be determined from existing literature augmented by additional laboratory polarization measurements which we will carry out at NIST during year 1. There will be trades between the need for speed, spectral coverage and precision. Liquid crystal modulators provide a flexible array of capabilities for beam referencing and switching, with timing capabilities well-matched to CCD readout speeds. In a field or astronomical application, improved spatial resolution improves discrimination of localized signals against the dilution of background mineralogical scenery. Hence, for our baseline configuration we envisage coupling polarization modulators to a robust, standard dual-beam imaging system with tunable wavelength. (We could, following the design studies, choose to emphasize spectral coverage, but the hardware component requirements would be very similar.) With an understanding of the levels of polarization introduced by realistic analogues of extraterrestrial life and with an understanding of the likely spectral characteristics, it will be possible to optimize studies of this form and to design instrumentation dedicated to the task. We propose to undertake a 3 month optical design study based on our derived requirements, followed by assembly of proto-type hardware intended to be useable in the field. The Confidential Page 52 2/12/2016 requirements and design phase will last approximately one year. Construction and testing of the instrumentation is expected to cover the second and third years, with enhancements and application for years three and four. Results will be published in the refereed literature. 5.3 Specular reflection from oceans on exoplanets Figure 5.5 Diurnal variations in reflectivity as the Earth’s terminator moves across its surface and the geometric location of the specular reflection point for the Sun crosses coastlines. We (CoI Turner working with Princeton graduate and undergraduate students) will further Specular reflection from the Earth’s oceans produces a substantial fraction of its total brightness and polarization; these can both change rapidly when the planet’s rotation causes the geometrical position corresponding to specular reflection to cross a coastline, as indicated in Figures 5.4. An analogous feature in the light curve of an exoplanet could provide an indicator of the presence of large bodies of liquid on the planet’s surface (Ford, Seager & Turner 2001; Seager, Ford & Turner 2002). In principle at least, a sufficiently extensive exoplanet light curve data set, ideally including polarization information also, could be used to construct a map of major land/sea divisions over some range of its latitudes (depending on the system viewing geometry)! The existence of large bodies of liquid, perhaps inferentially identifiable as water based on spectroscopic information, would obviously constitute a very important and provocative biosignature. We (CoI Turner working with Princeton graduate and undergraduate students) will further investigate this speculative possibility with an emphasis on its practical limitations and potential false positives. Using further simulations along the lines of those employed by Ford et al. (2001), we will seek to understand issues such as the systematic noise introduced by variable cloud cover, dilution of the specular reflection signal by other sources of polarization in the planet’s scattered light (e.g., atmospheric Rayleigh scattering), requirements for signal-to-noise Confidential Page 53 2/12/2016 ratios, light curve durations and sampling rates in order to reliably identify an exoplanet coastline, and so forth. A long-term goal would to develop automated techniques for coastline detection and/or mapping that could be applied to simulated or actual light curves of the Earth as a reference case for the method. The modeling work described here would also support attempts to detect the effect via the Earthshine observations described the following section. 5.4 Spectral signatures of land, ocean, weather and life At the present time Earth is the only planet known to harbor life, and studies of Earth are our starting point in characterizing any remotely detectable biosignatures. How would Earth appear, if seen from afar as an extrasolar planet? Could a mission like NASA's TPF determine if such a planet were habitable, or even inhabited? To what extent could we hope to learn about continents, weather patterns, and seasons? In this section, we propose tandem field projects focused on seeing Earth as a planet and exploring the dry limits of habitability in our own world. 5.4.1 Earth as an “Exoplanet”: Earthshine Over Time and Space Earthshine, light from the dayside Earth that can often be seen illuminating the dark side of the crescent Moon, contains the globally integrated spectrum of the sunlit Earth as it would be seen from afar as an extrasolar planet. In fact, Earthshine observation is currently the most accurate, inexpensive and easy way we have to monitor the spatially integrated spectrum of our own planet. While satellite spectra can provide useful information for small areas, summing such spectra does not accurately characterize the planet as a whole. In particular, the atmospheric path-length increases toward the limb, whereas satellites peer directly downward. Furthermore, satellite spectra are not taken with the full range of solar illumination angles, and large areas can only be captured over long time-periods. All of this dilutes changes we might expect to see due to weather and seasons. In previous work carried out separately by CoIs Turnbull, Turner and their respective collaborators (Seager et al. 2005; Woolf et al. 2002, Turnbull et al., 2005), Earthshine data were used to characterize the atmospheric, surface and biological signatures we might look for in an extrasolar planet spectrum. Rayleigh scattering, ozone and oxygen absorption, and atmospheric water vapor are the strongest signatures, and all are strongly suggestive of habitability; oxygen is suggestive of life itself. In some studies (e.g. Woolf et al, 2002, Tinetti et al, 2005), the spectral signature of vegetation was apparently present, but individual surface signatures are difficult to extract because the spectrum is spatially integrated over the entire planet. In order for distinct surface signals to emerge, we need to obtain spectra from many viewing angles of the planet. Spectrum-to-spectrum changes can be extracted at a higher level of significance then can unknown signals from a single spectrum. A major limitation of Earthshine studies conducted so far is that only two views of the planet are possible from a single observing site. The morning crescent Moon sees Earth to the east of our telescope, and the evening crescent Moon sees a view of Earth to the west (Seager et al. 2005). In order to capture other views of Earth, we must observe the Earthshine from other longitudes. Characterizing the Earth’s spectrum from many angles allows us to address two areas of astrobiological inquiry. First, how significantly would the spectrum of an Earth-like planet change as it rotates? This variation with longitude allows us to extract spatial information from Confidential Page 54 2/12/2016 a spatially unresolved signal. Second, how does the spectral signal change, as the view of Earth evolves from continent to ocean, from forest to desert, and from season to season? Or are these changes “masked” by the ever-changing landscape of clouds? Observing Earthshine over an extended time period will allow us to realistically assess the visibility of such signals for extrasolar planet discovery missions. To answer these questions, we propose a Portable Earthshine Monitoring System (PEMS). The essence of this project is simple: by transporting a small telescope and spectrometer to appropriate observing locations, we can spectrally characterize the entire planet in both space and time. Before describing this system in detail, we address an obvious question: Why use a portable system, instead of simply obtaining telescope time at various observatories around the world? Briefly, the reasons that a portable system is preferable are as follows: 1. For extended-source observations (such as the Moon), the important factor in obtaining high signal-to-noise is large beam size (i.e. low spatial resolution). Thus large (>1-m) telescopes (with high resolution and small beam size) are neither necessary nor desirable for Earthshine observations, 2. Small telescope (<1-m) observatories are not typically equipped with medium- to highresolution spectrographs as would be required for this program, 3. The quality of Earthshine data are strongly affected by slit-length and thus a single custommade instrument is needed (explained below), 4. Adapting a single, portable instrument to the diverse “back-end” interfaces of a suite of existing large telescopes would be time-consuming, awkward and expensive, 5. Local weather-patterns, which strongly affect data quality, can be avoided with a portable observing system, and 6. This project can be combined with the “Earth before Trees” project, thus achieving greater scientific return for essentially the same investment of travel time/costs and other resources. Turner and Wyithe have already acquired a portable Earthshine observing system that is based on commercially available equipment, specifically a Meade 203 mm LX200GPS-SMT SchmidtCassegrain telescope equipped with a Pictor 1616XTE CCD imaging camera and autoguider plus f/3.3 and f/6.3 focal reducers and a standard BGR filter wheel. The unusually large dynamic range of this camera and the use of neutral density filters permit us to obtain photometrically useful images of both the sunlit and Earthshine illuminated portions of the lunar surface with (separate) exposures of reasonable length. This system will be employed initially to search for the abrupt Earthshine brightness variability expected to be associated with coastline crossings of the Sun to Moon specular reflection point on the Earth’s surface (see Section 5.3). First observations will be obtained from the Mt. Arapilies area, west of Melbourne, Australia. For more general purpose Earthshine observations, we will add a spectrograph to the instrumentation Confidential Page 55 2/12/2016 package, ideally a long-slit spectrograph which allows simultaneous collection of data from the Earthshine illuminated portion of the lunar surface and the nearby background sky (in order to measure and subtract the scattered light from the much brighter sunlit portion of the Moon’s image). This may require the construction of a simple and portable special purpose spectrograph since most commercially available units suitable for mounting on a small telescope are fiber-fed and thus do not provide spatially distributed spectral data. In November-December 2005, Turner and Turnbull will visit their collaborators in Australia (S. Wyithe and his students at the University of Melbourne Physics Department) to test the existing observing system, establish a standardized observing protocol, and plan a 5-year observing campaign. The Earthshine observing experience of Turnbull, modeling experience of Turner, and instrumentation experience of Wyithe give this program a high probability of success. 5.4.2 Earth Before Trees: Globally-Integrated Spectra Ground-Truth Spectroscopy and Synthetic In tandem with the PEMS observing initiative (i.e., from the same field sites), we are beautifully equipped to carry out field studies that complement our study of the Earth as an “extrasolar” planet and as an “extreme” environment. In particular, there are two questions we can explore: How would the Earth appear as a “microbial” planet, without the existence of higher plants, as it was for the vast majority of its history? And how would the Earth appear as a “desert planet”, in either the case of much colder, or much hotter climate conditions? At the same time as our team is conducting Earthshine observations, we propose to spectrally characterize the Earth as it would appear in the absence of trees by obtaining ground spectra of (a) land areas that are dominated by microbial mats and (b) deserts. These spectra can be used in conjunction with software produced by the NAI Virtual Planetary Laboratory to create 3-D synthetic models of the desert Earth, and of the Earth without trees. These models will serve as interesting comparisons to the spectra generated with our “ground truth” data. Many of the un-vegetated sites that need to be visited for this program are also ideal sites for astronomical observation, by virtue of the fact that they are not prone to frequent precipitation or clouds. Thus, with one team traveling to selected field sites, we can efficiently carry out two missions, with one portable spectrometer pointing upwards and another pointed down. Again, before proceeding with the details of this project, we ask the obvious question: Why not use satellite data for desert regions and regions dominated by microbial mats? While satellite data can and will be a useful tool, those data are not sufficient for the following reasons: 1. Satellite data products are tailored to meet the needs of the satellite mission. Typically, this means that “spectral” coverage is really broadband imaging, with passbands chosen to elucidate specific phenomena (e.g., vegetation species, vegetation health, or minerals). Our goal is to cover the entire optical and near-IR spectral range at moderate resolution (R~600), focusing on different spectral signals generated by microbial and non-biological groundcover. 2. Satellite data products that are “hyperspectral” in nature (i.e., not broadband) are obtained at extremely high spatial resolution, covering very small, select land areas, and of limited Confidential Page 56 2/12/2016 wavelength coverage. Our goal is to spectrally characterize sites that cover a range of latitudes as well as the most extreme dry climate conditions, from the hottest to the coldest deserts on the planet, and containing surface microbes that inhabit polar to equatorial regions. 3. Hyperspectral data are not widely available, and the extraction of a data product that can be converted into a spatially integrated spectrum may require hiring special personnel to work with mission databases. Our goal is to carry this project out efficiently and in a manner where the data extraction procedures (and thus any artifacts) are clearly understood by the users. We propose to choose our PEMS field sites to be dually interesting for field spectroscopy of microbial groundcover and extreme desert conditions that are not expected to harbor extensive biological communities. Once ground spectra have been obtained, these ground truth data will be used, in conjunction with NAI VPL software, to create 3-D models of the “microbial Earth” and the “desert Earth”. These models, in comparison with our Earthshine Observations, will allow us to explore ways to spectrally distinguish between macrobiotic surface signatures, microbiotic surface signatures, and surfaces with little or no biological ground cover. The equipment required for this investigation is a simple field spectrometer, and for our purposes an off the shelf Ocean Optics Model S2000 spectrometer plus small laptop are entirely sufficient. Turnbull has prior experience with field spectrometers and will be the primary observer. 5.5 Solar-System bodies as exoplanet analogues Perhaps the most challenging aspect of pursuing an astrobiologically motivated study of an exoplanet will be extracting the relevant signal(s) out of the sparse flow of photons from its point image and arriving at a compelling, or at least plausible, interpretation of any possible biosignature(s). It seems likely that the endeavor will require a major research program involving many investigators, as diverse a suite of data as can be obtained and, no doubt, an extensive and controversy filled literature. In this context, it will surely be instructive and useful to prepare for these coming investigations of exoplanets by studying the relatively easy cases of Solar System bodies, for which something akin to “ground truth” information has been or eventually will be obtained by space probes. A particularly significant dimension of the problem that can be explored, in analog, via study of Solar System objects is the relation of spatially unresolved biosignatures to the complex underlying spatially differentiated properties of a target body. In this context, CoIs Turner and Chyba, working with Princeton graduate and undergraduate students, will carry out two projects combining ground-based observations with modeling studies. The former has extensive experience with optical observations with a wide variety of telescopes, instrumentation and targets, and the latter is similarly expert in modeling of bio- and geo- chemical processes on Solar System bodies. The ready availability of observing time on the APO 3.5-meter and its remote observing and fast instrument change capabilities make the proposed programs far more practical than it would be with more conventional large telescopes. 5.5.1 Martian methane Great excitement and considerable controversy has attended the apparent spectroscopic detection of methane in the Martian atmosphere at abundances far above expected equilibrium levels (Mumma et al. 2003; Krasnopolsky, Maillard & Owen 2004; Formisano et al. 2004) and with a Confidential Page 57 2/12/2016 distribution that appears to be spatially and perhaps temporally variable. Whether the source of this methane ultimately proves to be due to Martian life or abiogenic in nature, it seems sure to provide us with a powerful, and potentially sobering, lesson re the issues that will arise in interpreting biosignatures associated with exoplanets. Using the NIC-FPS (Near Infrared Camera - Fabry Perot Spectrograph) instrument available on the APO 3.5-meter telescope, we plan to monitor Martian methane via narrow band imaging centered on NIR methane bands, and adjacent control bands. These data should allow us to examine the spatial and temporal variability of any Martian methane signal over periods of weeks to months. Previous work on Martian methane has used non-imaging spectrographs and thus provides only limited information on the spatial distribution of the signal on the Martian surface, although there are claims that it is significantly inhomogeneous (Formisiano et al, 2004). If there are well localized, and potentially time variable, sources (venting) of the methane that produces the relatively diffuse signal seen spectroscopically, it will be much more easily detected via the high spatial resolution provided by the Fabry-Perot. Moreover, a synoptic monitoring program with a modest cadence (e.g., bimonthly during periods close to opposition) will provide a greatly increased chance of detecting episodic methane venting, whatever its source. And even a null result will provide a constraint on possible origins of the Martian methane. 5.5.2 Europa & Enceladus: ice planet prototypes If liquid water is a necessary condition for life, then ice-covered worlds with sub-surface oceans may well be the dominant reservoir for life beyond Earth (e.g., Chyba, 1997, and refs. therein). Within our own solar system, the volume of liquid water believed to exist below the ice and rock shells of the Gallilean satellites is 20-35 times greater than all of the liquid water on Earth (Spohn and Schubert, 2003). And one can add to this the recent, surprising, discovery by the Cassini probe of possible cryovolcanic activity on Saturn’s moon Enceladus. While icy worlds may be of direct importance for habitability, their spectral signatures may also serve as false positives for inhabited planets detected by missions such as the Terrestrial Planet Finder. Compounds typically associated with biological processes, such as O2 and O3, are known to exist on the surfaces of the Galilean satellites (Spencer and Calvin, 2002; Johnson et al., 2004), and yet we do not claim that these worlds are inhabited based on these spectroscopically intriguing results. Instead, we know that the interaction of the Jovian magnetic field with the surface ice results in radiolytic chemistry that produces a host of interesting oxidants. Such chemistry has been observed via spacecraft observations, space telescopes and ground based telescopes, and it is currently being studied in the laboratory environment (Vidal et al., 1997; Carlson et al., 1999; Moore and Hudson, 2000; Cooper et al., 2001). If missions aiming to discover habitable exoplanets are to be successful, they must be able to differentiate between non-biological planetary processes that can produce chemical disequilibrium (e.g. radiolysis or photolysis) and biological processes that are producing a true spectroscopic signature of life on another world. (Such radiation-driven disequilibrium could, in fact, be an important factor for the habitability of subsurface oceans on icy satellites (Chyba and Hand, 2001), but the radiation chemistry will occur whether there is an ocean under the ice or not.) Here we propose to address this issue through numerical models and ground based telescopic observations of icy worlds in our solar system. Confidential Page 58 2/12/2016 Detecting water from optical spectroscopy A reasonably thorough literature search reveals that very little modern, high resolution spectroscopy of these two bright objects (by the standards of large telescopes and CCD instruments) has been published and essentially none that explores a broad wavelength range, as opposed to concentrating only on some specific, pre-selected feature. The APO 3.5-meter Echelle yields a spectrum covering the entire CCD band (roughly 350-950 nm) at a resolution of 30-40 thousand in a single exposure; thus, the potential for serendipitous discovery via exploration of new domain of data is substantial. Using the currently available high dispersion echelle and later the NIR moderate dispersion TripleSpec (to be available within approximately two years) spectrographs on the APO 3.5-meter telescope, we will monitor these two moons to search for features indicative of their subsurface liquid environments, such as time variable, narrow water absorption lines. Gas phase water vapor escaping from such a moon’s surface should produce far narrower lines than surface water ice, and those features are potentially stronger and more variable than absorption due to any thin atmosphere produced by sputtering processes. The latter might also distinguish itself via correlation with leading vs trailing hemispheres. If such spectral indications can be identified, they will not only be useful for understanding the geologic activity on Europa and Enceladus on a longer time scale and with a better sampling rate than can be achieved with space probes but will, again, provide an example of what can be learned about such complicated objects based on limited and spatially unresolved data, one that will perhaps be instructive for interpreting the spectra of exoplanets. Observing Europa: the oxygen problem Understanding oxygen in Europa’s surface ice is important both to assessing the ways a variety of worlds could show signs of oxygen under remote observation, and to understanding the types and abundances of electron acceptors that could ultimately be available to support a possible biosphere in Europa’s ocean (Chyba and Hand, 2001). Condensed (gamma) phase oxygen has been proposed to explain the observation of the 5771 angstrom feature on the surface of Europa (Spencer & Calvin, 2002), yet it is known that solid O2 is not stable at average Europan surface temperatures (~100K, Johnson et al, 2005) and pressures ~10-6 µbar (Hall et al, 1995)). Radiolytic production of O2 is expected as a source, but laboratory measurements of this process have not fully clarified the process (Vidal et al, 1997; Moore and Hudson, 2000) and it seems unlikely that radiolytic production could offset atmospheric and ionospheric loss. Thus the observation of condensed oxygen presents a stability problem, requiring some form a trapping mechanism to retain the oxygen in the surface ice. Recently we proposed that the oxygen problem could be solved by invoking formation of a mixed SO2-CO2-O2 clathrate (Hand et al., 2006). Clathrates of these compounds are poorly studied and thus our identification of such material on other worlds is limited. Here we propose to use a field-deployable, visible, near-infrared, and mid-infrared Fourier transform spectrometer to study clathrates of CO2, SO2, CH4, O2, and N2. Raman spectroscopy and X-ray diffraction studies have been used in the past (Champagnon et al., 1997; Kuhs et al., 1997), but in order to remotely observe this ice phase with a spacecraft or space-telescope we must understand the spectral behavior in the visible, near-infrared, and mid-infrared. This suite of instruments has Confidential Page 59 2/12/2016 been successfully used in the Dry Valleys of Antarctica for analysis of both microbiology and mineralogy (Hand et al., 2005). We will bring this instrument to MBARI to study CH4, to the Polar Ice Core lab in Denver, CO, to study N2, O2, and CO2, clathrates in ice cores, and to Yellowstone Park during the winter in order to study naturally occurring SO2 clathrates around the sulfur-rich hot springs. Observing Europa: Radiation-Induced Planetary Fluorescence As the Jovian magnetic field sweeps past Europa with its 11-hour synodic period, the radiation impacting the trailing hemisphere of Europa will cause the surface ice to fluoresce in the UV and visible parts of the spectrum. During normal observations of Europa this fluorescence is overwhelmed by solar photons reflected from the surface. During an eclipse of Europa by Jupiter, however, such fluorescence could be detected. The orbital geometry required between the Earth, Jupiter, Europa, and the Sun makes such observations rare and difficult, but with dedicated telescope time at the Apache Point Observatory we propose to observe this effect over the course of the next five years. During December of 2005, nine such eclipses of Europa occur, but only four occur at night and all of the eclipses merge with occultations by Jupiter. The best opportunities during 2005 were in January and February, when the trailing hemisphere was visible during eclipse events that started far away from the disk of Jupiter (all ended with occultations, however) (Gillard and Holdaway, 2003). Thus, while the ideal conditions are rare, they are frequent enough to warrant a concerted effort to observe this phenomenon. The detection of this radiation-induced planetary fluorescence is important both for our understanding of processes that are occurring on Europa and for understanding possible radiation induced signatures from exoplanets. On Europa, radiolytic chemistry resulting from high-energy electron bombardment is believed to be occurring primarily on the trailing hemisphere. Understanding the global distribution of the radiolytic chemistry will aid us as we work to understand the role of exogenous and endogenous compounds on the surface of Europa and the consequences of such chemistry for life in the proposed sub-surface ocean (Paranicas et al., 2001; Carlson et al., 2005). Beyond our solar system, stellar and planetary magnetic fields will likely play a significant role in planetary surface and atmospheric chemistry; thus, understanding the signature of magnetically-trapped radiation on a moon in our Solar System may provide a useful context for future exoplanet studies. 5.6 Summary The principal tasks described in this section are as follows: We will validate circular polarimetry as a remote signature of life through measurements of terrestrial organisms and environments. We will use Earthshine observations to obtain an accurate characterization of potential spectroscopic biosignatures. We will acquire and analyze astronomical observations of Mars and the icy satellites, Europa and Enceladus, testing potential methods of exoplanet classification. Confidential Page 60 2/12/2016 6. Management Plan 6.1 Structure and organization Our project has a relatively simple management structure, illustrated in Figure 6.1. The PI, Neill Reid, has overall responsibility for the project. He will serve as the point of contact with external groups, including our Science Advisory Council, and the NASA Astrobiology Institute. NAI STScI Management Community Missions Office Executive Council PI (Reid) Institutional PIs (DasSarma, Turner) Program Manager SAC Science Advisory Council Exoplanet detection Exoplanet properties SNH Census Life and Cold Life and Radiation Mars & Europa New Biosignatures Kasdin Littman Vanderbei Paczynski Valenti Livio Turner Tremaine Sowers Robb Valenti Reid Turnbull Soderblom Postman Nota Paczynski DasSarma Sowers Robb Reid Chyba Robb DasSarma Chen Spergel Turnbull Chyba Turner Sparks Sowers Turner DasSarma Chen Turnbull Sparks Figure 7.1 The management structure of the NAI proposal team. The investigators are color-coded by institution: red, for STScI; green for COMB; and blue for Princeton. Our program involves three major partners: the lead institution STScI with the PI, 7 CoIs, and 4 collaborators; the Center of Marine Biotechnology (COMB), with 4 CoIs, 2 collaborators and two graduate students; and Princeton University, with 8 CoIs, 1 collaborator and four graduate students. Both STScI and COMB are located in Baltimore. Our project involves additional collaborators from a further 11 institutions, including Morgan State University, the University of New South Wales, Idaho State University, the Carnegie Institution of Washington, the University of Tokyo, the National Astronomical Observatory of Japan, the University of Melbourne, the Smithsonian Astrophysical Observatory, NIST, the Jet Propulsion Laboratory and Oxford Brookes University. Our collaborators include members of three other NAI teams (Carnegie, JPL/Caltech and Indiana). Prof. S. DasSarma will be the institutional PI for COMB, co-coordinating research, including contributions from collaborators, and serving as the main point of administrative contact, and Prof. E. Turner will perform the same functions for Princeton. The large number of investigators and institutions requires that considerable attention be paid to ensuring a true team effort that is both productive and cost effective for the scientific community Confidential Page 61 2/12/2016 and NASA. We have included specific labor in the proposal to provide oversight and coordination, which will take the form of a senior Program Manager at STScI. There are several highly qualified STScI managers with extensive experience in coordinating large and complicated efforts such as HST servicing missions, and large hardware and software development projects (our recently released Astronomer's Proposal Tool is a specific example). The Program Manager will be devoted to the NAI program at 0.3 FTE per year. S/he will work closely with the PI to establish the schedule (including milestones) and co-ordinate with COMB and Princeton University work and deliverables. The Program Manager will also ensure rapid disbursement of funds to the subcontracted institutions, Princeton and COMB. The PI and the two institutional PIs, together with the Program Manager, will form the Executive Council of this project. They will collectively decide outstanding issues involving intellectual property rights and publication policy, and they will adjudicate should any disputes arise. Should the Executive Council fail to reach agreement, the final decision rests with the PI. Coordination within the science team requires good communications. All three institutions have existing telecon and video conferencing facilities that will be used for this purpose. In addition, STScI has pioneered the technique of web casting and archiving, which will be utilized to provide widespread access to and permanent records of our activities. We have a great deal of experience working as part of a large and widely dispersed team to tackle challenging projects. We anticipate regular (weekly) internal meetings at STScI, COMB and Princeton to review progress. We will hold bi-weekly team videocons to review overall progress and enable good communication and cross-disciplinary discussion among the broader team. We will organize a Workshop on Astrobiology and Extremophiles in years 1, 3 and 5; COMB faculty and staff will take the lead in planning and execution. In the intervening years we plan to have 3 to 4-day retreats for the full team, to foster interaction between different research projects. At STScI, the NAI project will fit seamlessly into the existing management structure as part of the Community Missions Office (CMO), which currently oversees several externally funded projects that are independent of our prime contracts for the Science Operation Centers to operate the HST and JWST, including MAST ($1M), and STScI’s involvement in the Kepler Discovery mission ($7.5M) and the National Virtual Observatory ($2M). The internal STScI management oversight would include a monthly CMO status meeting, a written monthly summary of project status, and a quarterly oral presentation on the status of the project to STScI’s senior management team. Existing administrative and management personnel are already in place to support this project. This provides our team with unique management assets that are efficiently leveraged from a large existing experience base. It is our view that a project of this scope will also benefit greatly from a completely independent external Science Advisory Council (SAC) convened annually to provide a thorough review of, first, the plan, and then the progress of the project. This group will be a team of 5 astronomers, planetary scientists, geologists or biologists, selected by the Executive Council, who will provide advice on on-going activities and on relative priorities within the project. We anticipate that individual members of the SAC will serve 2-3 years. Confidential Page 62 2/12/2016 6.2 Schedule: Milestones and deliverables Table 6.1 shows the major milestones and deliverables for the main research projects that are part of our NAI proposal. SNH Census HighCold life Radiation Exoplanet New contrast & life properties biosignatures imaging Construct v1.0 Complete Initiate cold Initiate M- Design field Year 1 database and populate with stellar data Year 2 Release v1.0 SNHC to team members for additional input Implement initial habitability criteria Identify and acquire key missing data Year 3 Update and expand SNH Census to v2.0 Release to NAI community for additional input. Incorporate results from dynamical models Continue to acquire key missing data Update SNH Census with initial results from Kepler Update habitability criteria Year 4 Year 5 Release v3.0 of SNH Census Confidential TPF-C mask designs Design Subaru masks Begin analysis of speckle algorithms Complete analysis of close-loop amplitude control Manufacture Subaru pupil masks Begin optical design if HiCIAO coronagraph module Deliver pupil designs for ground-based instruments Propose for funding for HiCIAO coronagraph & salt, cold & anoxia, and cold and CO experiments dwarf flare monitoring in open clusters Initiate Phase II planetary system models Continue cold & salt, cold & anoxia, and cold and CO experiments Initiate biofilm and gene transfer studies Continue Mdwarf flare monitoring Initiate Mdwarf ageactivity program Continue Phase II planetary system modeling Continue low temp. growth, biofilm and gene transfer studies Initiate ecological and proteomics studies Initiate extremophile radiation sensitivity experiments Initiate Martian soil experiments Manufacture and deliver pupil plane coronagraph for HiCIAO Complete biofilm and gene transfer studies Complete ecological/ proteomics studies Continue extremophile radiation sensitivity & Mars soil experiments Ground-based observations of SNHC targets with HiCIAO Complete radiation sensitivity & Mars soil experiments Page 63 Continue Mdwarf flare monitoring in open clusters Continue ageactivity program Complete Phase II planetary system models Initiate `small planet’ models Complete Mdwarf flare monitoring Complete ageactivity program Continue `small planets’ modeling Complete `small planets’ program circular polarimeter Construct traveling Earthshine system and make first observations Start construction of field polarimeter Continue Earthshine measurements Complete construction of field polarimeter, start laboratory test measurements Continue Earthshine measurements In situ measurements with field polarimeter Complete Earthshine measurements Complete field measurements with polarimeter 2/12/2016 6.3 Key personnel Dr. I. N. Reid (STScI) is the Principal Investigator. He has overall responsibility for the project and will serve as the point of contact with external groups. He has conducted research on nearby low-mass stars and brown dwarfs for over two decades, and has recently applied his expertise in Galactic structure to investigations of the properties of exoplanet host stars. Prof. S. DasSarma (COMB) is the institutional PI for the Center of Marine Biotechnology. He will be responsible for co-coordinating the biological experiments undertaken at COMB. He is a pioneer in the study of halophilic Archaea and a well-known expert on microbial genomics, transcriptomics, and bioinformatics. He is leading several key experiments that will provide insights into adaptation of halophiles to low temperatures and resistance to damaging radiation, in collaboration with DeVeaux, and McCready. Prof. E. Turner (Princeton University) is the institutional PI for Princeton and will be responsible for coordinating the astrophysics and engineering projects undertaken there. He is a widely experienced optical astronomer who will work on innovative ground based observations of exoplanets, Earthshine and Solar System objects. He has also contributed to the investigation of potential exoplanet biosignatures. Prof F. Chen is an expert on marine cyanobacteria and cyanophage, and will be responsible for testing the cold and radiation tolerance of cyanobacteria isolated from various sources, including some samples collected from Antarctic. Dr. C. Christian is highly experienced in scientific outreach, and has pioneered the use of information technology for educational and research activities. She will co-ordinate the E/PO activities of this project. Prof. C. Chyba is widely recognized as an intellectual leader in the field of astrobiology and has made significant contributions on a wide variety of topics, ranging from SETI to cometary delivery of volatiles to objects in the inner Solar System. He is an expert on possible Europan and Martian biology, which will be the foci of his efforts supported by this project. Prof. N. J. Kasdin is an expert on systems engineering and advanced optical technology and instrument design. He leads the Princeton high-contrast coronagraphy group and will coordinate its activities internally and in interactions with the rest of the team. Prof. M. Littman is an optics and robotics expert who will have primary responsibility for laboratory optics work at Princeton. Dr. M. Livio has an extensive resume of past work on galactic, stellar and planetary astronomy. He will provide input primarily on Galactic habitability issues, and will also participate in the outreach program. Dr. A. Nota has extensive expertise on stellar evolution, and will be a key science contact in developing the Solar Neighborhood Habitability Census. Prof. B. Paczynski is a pioneer of the study of Galactic microlensing and of its application as a technique for detecting exoplanets and is the intellectual leader of the OGLE project. Working with Princeton graduate students he will use OGLE data to study the statistical properties of the exoplanet population in the Galaxy Confidential Page 64 2/12/2016 Dr. M. Postman will provide project management oversight. He has extensive experience in database construction, and will participate in construction of the SNH Census. Prof. F. Robb is an expert on Archaea and thermophilic life. He will lead the study of microbial survival on minimal energy and carbon sources, during radiation challenges and under cyclic temperature variations. He and collaborators Onstott, Steele and Fogel will study microbial enrichments from Arctic and subarctic Mars analog sites. Dr. D. Soderblom has extensive expertise on nearby stars, particularly chromospheric and coronal activity, and will be a key science contact in developing the Solar Neighborhood Habitability Census. Prof. K. Sowers is an expert on the physiology and molecular biology of the methanogenic Archaea. He will be responsible for all experiments proposed with for the Antarctic methanogens. He is also a collaborator for the genome sequencing projects for both psychrotolerant methanogens and will collaborate with R. Cavicchioli, a leader in proteomics of the psychrophilic Archaea, to investigate the molecular mechanisms of cold adaptation Dr. W. Sparks is an expert on elliptical galaxies. He is currently investigating the potential of circular polarization as an unambiguous biosignature; he will lead work on the development of the field polarimeter. Prof. D. Spergel is one of the country’s leading theoretical astrophysicists and has worked on an uncommonly broad range of topics, ranging from early universe particle cosmology through Galactic dynamics to advanced optical theory. He will work on both optics and theoretical issues related to panspermia in this project. Prof. S. Tremaine is an expert in theoretical dynamics and has worked extensively on problems of Solar and exoplanetary system dynamics as well as planet formation theory, the areas of his involvement in this project. Dr. M. Turnbull is currently an NAI Fellow at the Department of Terrestrial Magnetism of the Carnegie Institution of Washington. She has extensive experience in analyzing planetary habitability, and on measuring Earthshine. She will be based at STScI and will work on both the Earthshine project and work building and testing the Solar Neighborhood Habitability Census. Dr. J. Valenti is an expert in stellar activity and stellar chemical abundance analysis. He will also provide information on the detailed properties of the host stars of known extrasolar planetary systems. Prof. R. Vanderbei is an expert in numerical optimization and optical designs who has made fundamental contributions to the theory of high-contrast coronagraphs. He will work on these topics as a part of the coronagraph work at Princeton. Confidential Page 65 2/12/2016 7. Strengthening the NAI Community E/PO Professional community development Training Minority institutions Staff participation Facilities Flight missions Information technology 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 8.1 8.2 8.3 8.4 8.5 8.6 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 7.1 Strengthening the Astronomical Connection Our astronomical team members bring to the NAI a wide range of expertise, both technical and scientific, in astrophysics and space sciences. Our team includes experts on cosmology, stellar evolution, stellar populations, Galactic structure, extrasolar planets, planetary formation and dynamics, brown dwarf and planetary atmospheres, planetary environments and Solar System objects, including Europa, Titan and Mars. In particular, several CoIs are involved in research covering a broad span of techniques for detecting extrasolar planets. This diversity is essential for understanding the Galactic landscape, the first milestone on the Astrobiology Roadmap. The Space Telescope Science Institute is one of the most prestigious astrophysical institutes in the United States and is renowned for its public outreach efforts related to the Hubble Space Telescope. A recent review of the NASA Astrobiology Institute by the Committee on the Origin and Evolution of Life (COEL) of the Space Studies Board highlighted STScI as a potential catalyst for creating an astronomical influence in astrobiology. Through its role in HST, STScI has close contacts with the astronomical community, and it has used those contacts to engage the community in astrobiological issues, notably through the 2002 (`Astrophysics of Life’) and 2005 (`A Decade of Extrasolar Planets’) May symposia. Princeton University has a long and storied involvement in astrophysics. In recent years, Princeton was one of the prime movers behind the Sloan Digital Sky Survey, surveying steradians of the high latitude sky. Princeton is closely involved in technical and scientific developments in planet detection, and is currently establishing a Center for Planets and Life. The University is also in the process of developing a certificate program in astrobiology that will be offered to undergraduate students. Confidential Page 66 2/12/2016 7.2 The Biology Connection The Center of Marine Biotechnology, one of five research centers of the University of Maryland Biotechnology Institute, conducts biotechnology research in the context of the marine environment; provides training and public outreach to various local, state, national, and international audiences; and encourages economic development within the State of Maryland. Housed in modern state-of-the-art laboratory facilities at the Columbus Center in Baltimore’s Inner Harbor, COMB is conveniently located within a short driving distance of STScI. COMB’s mission is to apply the modern tools of biology and biotechnology to study, protect and enhance marine and estuarine resources. Among the 40 members of the COMB faculty is an outstanding group of investigators with expertise in a wide range of extremophile biology. With extensive research publication records in extremophile genomics, physiology, biochemistry, and molecular biology, these laboratories cover the lifestyle spectrum of extremophiles, life in extreme heat and cold, high ionic strength, and in oxygen-deprived environments and play a leading role internationally. As co-editors on the Cold Spring Harbor Laboratory Press Handbook, Archaea: A Laboratory Manual, they have sponsored annual symposia and workshops on extremophile biology. As a marine research institution, COMB is fortunate to partner with Maryland SeaGrant in a variety of education and outreach activities ranging from curriculum development to teacher training and school-based programs. CoIs DasSarma, Sowers and others are supported through the NSF and DOE Microbial Genome Programs, while Robb, Chen and other members of the COMB faculty are funded to be participants in the Microbial Observatory Program. Together with collaborators like Andrew Steele and Marilyn Fogel at the Carnegie Institution of Washington and Richardo Cavicchioli at UNSW, they have access to a wide variety of extremophilic species and genome sequences suitable for analysis as part of our astrobiology program. Over the last two years, COMB and STScI personnel have been engaged in active scientific collaboration, placing the biological investigations in an astronomical context. The results of these experiments prompted some of the research outlined in the present proposal (see section 4.2.4), and they have been submitted for publication in the journal Astrobiology. 7.3 Communicating astrobiology in the research community While there remains some skepticism about Astrobiology in the Astronomical and Biological communities, we are committed to bridging these “culture” gaps through focused outreach at professional meetings in our respective home fields. For astronomers, the most significant meetings are the American Astronomical Society winter meetings, and as an NAI team we will engage the AAS organizers in creating an “Astrophysics of Life” symposium. This symposium will be lead by early career and senior astronomers (including PI Reid and CoI’s Turnbull, Valenti and Livio) who will introduce their colleagues to the outstanding questions and latest findings in Astrobiology. The American Society for Microbiology has been and will continue to be a valuable vehicle for communicating the importance of astrobiology. At the 2005 ASM General meeting in Atlanta, CoI DasSarma gave a symposium lecture on astrobiology; at the 2006 Orlando meeting, he will serve as co-convener on an Astrobiology session and involving other members of the NAI team. Confidential Page 67 2/12/2016 We are intent on developing a genuine synergy between the biological and astronomical scientists and as we described in our research plan, as a step towards that symbiosis we are producing a compendium of scientific reviews, archived webcast presentations, and explanatory articles to enhance our own research efforts. This material will be hosted at STScI and available from the NAI web pages, and will greatly enhance the accessibility of our research to other members of the NAI and the general scientific community. We intend to continue to foster regional interest in astrobiology by organizing further workshops, hosted at STScI, COMB and Princeton. Those meetings will cover a number of astrobiology topics, including the biological impact of changing planetary environments, mapping life through the Galaxy and novel techniques for extrasolar planet detection. 7.4 Minority Institutions Collaborator Jochen Müller (formerly at COMB) has recently taken up a faculty position at Morgan State University, Baltimore. We anticipate that undergraduates from that institution will participate in some of the biological experiments outlined in this proposal. 7.5 Undergraduate Astrobiology Training Exposure to astrobiology at the undergraduate level can significantly enhance the integrity of the Astrobiology community by building a base of enthusiasm among the general public and by recruiting future Astrobiology researchers. At Princeton, a group of 15 faculty members from 8 science and engineering departments at Princeton are engaged in an ongoing series of educational initiatives intended to establish a major international center of activity in the intensely interdisciplinary field of astrobiology. These include: 1. Teaching a “sophomore initiative” cross-listed course (GEO/AST/EEB/CHM 255: Life in the Universe), currently being taught by Profs. E. Turner, C. Dismukes, L. Landwebber, T.C. Onstott and E.I. Steifel. 2. Developing a certificate program in astrobiology, a proposal currently under consideration by the University 3. 3. Supporting a new undergraduate organization (The Princeton Astrobiology Club, P-ABC) recently established via student initiative. The major educational component of this suite of initiatives is the Astrobiology Certification Program (ACP). If approved, it will allow Princeton undergraduates to formally add a “Certificate in Astrobiology” to their departmental concentration (major) on their diplomas by: 1. taking a special two semester astrobiology sequence (typically in their sophomore years), 2. completing relevant junior and senior year independent work projects on topics approved by the ACP director and the student’s department of concentration, 3. taking four “cognate” courses (regular courses deemed important to Astrobiology, e.g. biochemistry, biophysics, evolutionary biology, petrology, astronomy), two of which may be in the student’s home department, and 4. participating in a senior thesis colloquium. Confidential Page 68 2/12/2016 The undergraduate program at Princeton will be augmented by a Joint Astrobiology Student Internship Program connected to STScI and COMB under the mentorship of CoIs Valenti, Turnbull and Robb. In a cooperative program, summer students (some of which we hope will be recruited from the Princeton ACP) will have the opportunity to interact with one another at all institutions. Students will work individually with mentors from each institution and they will convene for weekly meetings, workshops and social activities at alternating locations between STScI and COMB. In this way the students will have the opportunity to: 1. work closely with one mentor pursuing their own research project, 2. interact and socialize with other students at both their “home” institutions and in our astrobiology consortium, and 3. gain cutting-edge cross-disciplinary exposure to the broad range of research done at STScI and COMB STScI and COMB will each support two interns; Princeton will support one student from their regular summer internship programs. 7.6 Graduate and postdoctoral astrobiology training: The COMB Extremophile Summer School One of the NAI’s unique roles in the scientific community is that of providing scientists, especially those in the early stages of their careers, opportunities to gain Ph.D.-level experience in new disciplines. The STScI-COMB-Princeton consortium, in particular, can offer graduate students, postdoctoral fellows, and other researchers a special opportunity to work first hand as biologists, with a curriculum that is tailored to those who come from non-biology backgrounds. We propose to host a “Extremophiles in Astrobiology: Theory and Techniques” (EATT) fiveday summer school at the COMB facility, open to graduate students, postdocs, and other researchers who engage primarily in “non-biology” astrobiology research. The EATT Workshop is intended to complement workshops held at other NAI institutions (e.g., the University of Hawaii and the University of Arizona) that are intended to help life-science and Earth-science students become more fluent in the languages of planetary- and space-sciences. “What would you do if someone handed you a bag of rocks from Mars?” While most astrobiologists who work in the fields of atmospheric, geological, planetary, or space science would love to know whether samples collected (or investigated in situ) on other planets contain biological activity, very few would have any idea where to begin with such a question. Indeed, most scientists who were not “raised” as biologists have a difficult time understanding the language of biologists and the techniques they use to characterize microbial life on Earth, and this adds to the struggle in keeping up with exobiology research. Getting a handle on which techniques provide what information and are subject to which limitations is several steps beyond the biology expertise of most space/planetary science Ph.D.’s. The EATT summer school is designed to start filling in these gaps of understanding in an engaging environment that fosters new relationships among participants. At the EATT summer school, participants will engage in: (1) hands-on biology lab experience, (2) seminar-discussion sessions focused on extremophile research and pitched to the level of a non-expert audience, and (3) the opportunity to see and use COMB’s unique facilities for the study of anaerobic biochemistry and molecular genetics. Confidential Page 69 2/12/2016 To create a summer school program that is appropriate for NAI and other astrobiology scientists, we have taken the highly successful curriculum that our COMB CoIs DasSarma, Sowers and Robb developed, used, and refined over the last decade, and adapted it for an interdisciplinary Ph.D.-level audience. The course description and curriculum are as follows: EATT: Objectives The “Extremophiles in Astrobiology: Theory and Techniques” course will be held during the summer at the University of Maryland Biotechnology Institute’s (UMBI) Center of Marine Biotechnology (COMB) teaching labs in the Columbus Center, Baltimore Maryland. This course, which includes a one-and-a-half-day symposium, three days of intensive laboratory experience, and an afternoon for students to share their own research through poster presentations, will be administered by the Office of Continuing and Extended Education, University of Maryland College Park in association with COMB. The symposium will bring together experts from astronomy and from various areas of extremophile research to discuss the current state of knowledge of growth, biochemistry, genetics, ecology and diversity of microorganisms that thrive in unique environments, and to place that research in an astrobiology context. Taking advantage of the expertise developed at STScI, we envisage that at least a portion of the symposium will be webcast. The laboratory sessions include lectures and blocks of demonstration and hands-on experiments focusing on specialized techniques used to study these microorganisms. EATT: Workshop Schedule Our NAI consortium’s “Extremophiles and Astrobiology: Theory and Techniques” has been adapted from a summer course offered each year at COMB since the summer of 1997. We envisage the course lasting a week, with an all-day symposium on the opening Monday, and on the Friday morning, with the afternoon given over to student poster presentations. The central part of the week is devoted to hands-on laboratory experiments. The first-day symposium will be introduced with a presentations and discussions that provide, from a space scientist’s perspective, the astrobiological context of extremophile research. The symposium talks will generally be 50-55 minutes in length, with 5 minutes for questions. Invited speakers will be drawn from the general community, but we anticipate that both STScI and Princeton will contribute speakers on the “astro” aspects of astrobiology. Each speaker devotes 30 minutes to general aspects of their research area with the remaining time dedicated to their own research specialty. Reid, Turner, and Turnbull will work specifically to make sure that all of the material presented to students is accessible and relevant to those who are building careers in non-biological sciences. In the experimental section of the workshop, each day is divided into three 2.5-hour modules, one each for halophiles, methanogens and thermophiles, allowing participants to be divided into four groups of three to four. The groups then rotate through the modules. The faculty presiding over the modules are experts in their particular field and are assisted by graduate students and/or postdoctoral students who work in their laboratories. Confidential Page 70 2/12/2016 With specialized facilities for carrying out extremophile research, COMB is the ideal location for this workshop. COMB houses dedicated anaerobic laboratories that are specially equipped with instruments for conducting both small and large-scale batch and continuous culturing of strict anaerobes as well as biochemistry and molecular genetics under strictly anaerobic conditions. The laboratories, including the two teaching laboratories used in this course, are plumbed for the distribution and mixing of anaerobic gasses at several bench stations. The facility also contains apparatuses for preparing anaerobic media, anaerobic gassing manifolds, and Coy anaerobic chambers: one chamber equipped for anaerobic microbiology and the other refrigerated and equipped with a dedicated HPLC for anaerobic biochemical separations. COMB also houses a unique fermentation facility, the Extremophile Scale-Up Facility (ExSUF) that is equipped with a customized pilot-scale 250-liter fermentor and 20-liter seed fermentors adapted for growth of strict anaerobes and hyperthermophilic microorganisms at temperatures up to 105°C. The support we are requesting to fund this workshop includes costs associated with about 100 participants for the “theory” portion, and 16 students for the hands-on “techniques” segment. Funds are requested for laboratory supplies, our course textbook (Archaea: A Laboratory Manual published by Cold Spring Harbor Press), logistical costs (for the symposia and field work), and instructor and student travel and lodgings support. As discussed in detail in budget narrative, we are requesting a total budget of $40,000 per workshop, and we anticipate holding workshops in years 1, 3 and 5. EATT: Target Audience The ERTT workshop is intended for advanced graduate students, postdocs and other researchers who have begun their own research program in a non-biology-focused, but astrobiology relevant, field. Early career participants will be given priority for attendance and financial support, but researchers at all stages of their careers will be invited to attend provided there is room. Funding will be provided only to graduate students and postdocs, and all funded participants will be expected to contribute a poster in our afternoon poster session. We envisage the experimental section of the workshop accommodating up to 15 participants. EATT: Promotion Marketing for the workshop will be done via NAI electronic newsletters, print media announcements, announcements and fliers at the Astrobiology Graduate Conference, the Astrobiology Science Conference, the Division of Planetary Science meeting, the American Geophysical Union, and other space, planetary and geological science meetings, as well as meetings of the American Society of Microbiology. Turnbull will also distribute information via the early career astrobiology email list started at the first Astrobiology Graduate Conference sponsored by the NAI and via the Ames Academy Alumni listserv. Finally, fliers will be distributed to NAI nodes for posting. 7.7 Astrobiology Postdoctoral and Graduate Fellows Our proposed research program will also involve the direct participation of at least 6 graduate students and 5 postdoctoral fellows. Most of those personnel will work either at COMB, on biological experiments, or at Princeton University. We anticipate that two postdoctoral fellows Confidential Page 71 2/12/2016 will based at STScI, working on developing the SNH Census and on the construction and implementation of the circular polarization field instrument. We also will take advantage of the summer student program run by STScI to involve undergraduate students in astrobiology research. Princeton University faculty members participating in this proposal will incorporate aspects of this research and astrobiological concepts in their undergraduate and graduate courses, and CoIs from COMB/University of Maryland are actively involved in teaching graduate and undergraduate courses covering a range of levels, and topics from ecology to bioinformatics. We anticipate that our research tools will be used in a number of those courses. 7.8 Astrobiology and relevant NASA Flight Missions The research program outlined in our proposal is directly relevant to several of the most prominent space missions currently in preparation by both NASA and ESA. Those include KEPLER, SIM Planetquest, TPF, ESA’s DARWIN mission and, eventually, Lifefinder. Our team members have extensive involvement in those projects: Collaborator Gilliland is a CoI in the KEPLER project; PI Reid is a member of two SIM Planetquest Key project teams; and CoI Sparks was a member of the TPF Science Working Group. All of these missions are directed towards identifying and studying extrasolar planetary systems. As discussed in our proposal, KEPLER is a wide-field imaging instrument that will search for transiting planetary systems. The results from that project will provide significant new information on the frequency and characteristics of sub-Jovian mass gas giants and terrestrial planets. In contrast, SIM, TPF and DARWIN are all pointed missions, designed to target a relatively small number of sources. This is particularly the case for TPF and DARWIN, missions that will devote a substantial fraction of their total program effort to observations of ~100 stars within the immediate Solar Neighborhood (d < 20 parsecs). Clearly, it is vital to select the best possible targets for those programs. The research program outlined in this proposal will provide a quantitative framework for addressing exactly this issue. The Solar Neighborhood Habitability Census will catalogue both empirical data and the derived intrinsic properties for stellar systems near the Sun, including those stars that will be considered candidates for detailed study by TPF and DARWIN. We will be able to use the statistical tools created as part of our research program to combine relevant information to yield a ranked list of nearby systems most likely to host life. Some systems have known extra-solar planets in the habitable zone, while others have companions that preclude stable planetary orbits in any habitable zone. The remaining stars will not be perfectly characterized, but we will still be able to rank them on the basis of statistical trends observed in system with known extra-solar planets. The optimal biomarker to use when searching for life will depend on the expected environment for each system being studied. All team members at STScI and many of the Princeton CoIs have extensive experience with NASA flight missions. Most STScI personnel are involved on a daily basis with operation of the HST, and have participated in HST servicing missions and the development cycle of new instruments. Some are already working daily on the James Webb Space Telescope, which will be an important tool for astrobiology. Our experience with current and future NASA flight missions will naturally guide the focus of the research tools we develop in this project, making those tools more useful for planning and executing future NASA flight missions. Confidential Page 72 2/12/2016 8. Education, Outreach and Press Presence 8.1 Capturing public imagination: Education, Outreach and Public Understanding of Science Imagine sitting before a home computer and using a pair of affordable stereo vision goggles, calling up an image of a starfield. Suppose you could fly about the scene to see a stereo rendering of the various components, navigating around stars, star forming regions and dark areas of hydrogen and dust. Highlighted areas point out planetary systems in various stages of formation. Zooming in on a particular region, you see a star system being born. Perhaps you are interested in calling up the detailed explanation of one of the planets in that system that caught your eye. You click a button, or perhaps issue a voice command and a description of the planet appears, superimposed on the view like an heads-up display. What kind of microbes might live in that environment? Lets find out! The public as well as students and teachers will discover with us the types of planetary systems that are known and how to detect new systems through emerging observational techniques. Our public audiences will be able to characterize the nature of exoplanetary systems found and speculate on the types of environments present in those systems. We will investigate the variety of conditions for survivability and adaptability of potential life forms using our research on extremophiles as a guide. By putting the two together, that is, the astrophysical and biological analyses, our public explorers will be able to experience for themselves the possibilities for habitable environments in the Solar Neighborhood. Our multifaceted outreach program will cultivate exploration and study by collaborating with existing programs both within the NAI and the general science education community. We acknowledge that the fundamental questions embedded in astrobiology arouse the imagination – How did life arise? Are we alone? What is our future? We will use that fascination as a springboard for our education and outreach activities. We will employ the tools of instructional technology -- the Web, the Internet, and the media coupled with existing educational resources and networks to create a direct access to the investigations. We will approach Education, Outreach and Public Understanding of Science (referred to within this text as EPO) through several complementary activities. We will create our Astrobiology EPO program to take full advantage of the long established education and public infrastructure experience and infrastructure of STScI’s Office of Public Outreach (OPO), COMB’s expertise in teacher research collaborations and professional development and Princeton’s success with astrobiology graduate and undergraduate education. We understand that to be most effective, our program cannot be built from “scratch”, especially given the limited funding allowable for this proposal. We also have been selective about which areas of EPO we will pursue. We will collaborate with the skilled and expert staff of OPO, use existing networks, and distribution channels as well as link to related resources, background material and curriculum materials to maximize the impact of our efforts. We already have ties to several groups within the NASA Astrobiology education community through the NASA Origins Forum, managed by STScI/OPO. Our spectrum of activities (Table 8.1) will be well coordinated with the NAI and NASA Confidential Page 73 2/12/2016 Education Support Network. In this EPO section we discuss up through K-14 education. The college level education is shown in the Table for completeness, is discussed elsewhere in this proposal, and emphasizes Princeton’s role in undergraduate and graduate astrobiology education. In brief, our “ExtremoFiles: Notebook” will highlight the progress of our research along with salient updates, news notes and occasionally, press releases as our research progresses, and conduct experiments in the biological laboratories. This area also includes “Kids News” from the Amazing Space suite of resources. “ExtremoFile Explorer” is a collaborative project with the National Virtual Observatory (NVO), OPO, and ManyOne Networks to create two-dimensional imagery and three-dimensional renderings of our local solar neighborhood from astronomical survey data available in the NVO such as the Sloan Digital Sky Survey, other sources, and our own Habitability Census data resulting from the research in this proposal. We will represent the stellar population attributes down to the local elements containing planetary systems and the potential for hosting microbial organisms. The 2-D imagery will be folded into modules usable for planetarium presentations, science museum kiosks and Web-based public resources where the public can join us in our explorations. We also will sponsor lectures open to the public at our sites and at museums and broadcast via webcast technology over the internet. Table 8.1 Spectrum of EPO Activities, described in detail in the following sections. Type of Outreach News Public Information/ Outreach Informal Education K-14 support Science curriculum College/ University curriculum Element ExtremoFile Notebook Typical Products News releases Media contacts interviews Web resources Linked resources Paper products Self-guided modules Content News text, images, mediated & / derived products, multimedia, biographical sketches ExtremoFile Mediated products, images, lab Explorer notes, 2-D and 3-D solar neighborhood model hypertext linked to exoplanet characteristics ExtremoFile Exhibits Images, models, laboratory Explorer, Planetarium shows materials, 2-D and 3-D solar ViewSpace Kiosks neighborhood models hypertext linked to exoplanet characteristics Laboratory, Links to Curriculum Images, processed data Teacher modules, Self-guided products, science data and Research modules parameters, models, hands-on Fellowships, Teacher Workshop laboratories Living in the Teacher Professional Extreme Development Astrobiology Courseware Images, science data, processed Certificate Self-guided modules data products Projects Relevant formal education curriculum support tools already developed for use in classrooms at STScI and COMB will be linked to our program through our “ExtremoFile Laboratory Researcher” initiative. The educational resources are in use nationally and reach a significant Confidential Page 74 2/12/2016 segment of underrepresented and home-schooled students in all 50 states. All products developed for classroom use are linked to national science, technology engineering or math standards and Project 2061 Benchmarks, integrated into ongoing NASA OSS education activities and subjected to rigorous review and formative evaluation processes. We will disseminate late-breaking discoveries into the classroom through established outlets at NASA, NAI, STScI and COMB. Each of these areas is discussed in detail below. In the descriptions we articulate our commitment to wed the EPO activities directly to the research being conducted and the investigators who drive the program. 8.2 ExtremoFiles: Notebook Communicating with the Public. We know there is tremendous public interest in the origin of life and the tantalizing possibility that life exists elsewhere in the solar system and galaxy. We will capitalize on this curiosity and stimulate the public interest in our Astrobiology project by communicating the latest information on our studies to the public and educators through our many faceted project website. We will create a window into our Laboratory (a virtual astrobiology visitor center) to showcase the progress of our experiments and our models of the solar neighborhood. Through vignettes into our scientific “laboratory notebooks” we will describe online, using current technologies such as weblogs, RSS feeds and the like (see Figure A), the context of our work, our strategies and the latest results. We will illustrate our Notebook with simple graphical representations of our models, imagery, straightforward explanations and background material covering the nature of stars, galaxies, planetary systems and adaptive biological organisms. This part of our project website will be developed by the Office of Public Outreach (OPO) at STScI. Alerting the Public through the News Media. We occasionally will prepare and distribute press releases with the News Group of OPO, a group that has excellent professional rapport with the media and a long history of accurately communicating space science to the press. As is our natural practice at STScI, we will coordinate with the home institutions of our collaborators and in this case, also the experience and leverage of the NAI. All press materials will also be available electronically through the Notebook portion of our website as well as linked through OPO, NASA (as appropriate) and our collaborating institution. We will include a “kids news” area based on OPO’s Star Witness feature (amazing-space.stsci.edu/news/). The content provides online reading selections mirroring the content of press releases, but for grade school students to enjoy. Teachers can elect to address reading concepts using passages that expose the students to new science ideas. We also will offer live interviews, Q&A sessions, animation, graphics, textual information and imagery captured and rebroadcast on the Web for use in schools, libraries, science museums and by the interested public. Major scientific findings (anticipated to be a few over the course of this project) will be showcased through NASA press conferences, the Space Science Updates, produced by STScI and broadcast from NASA headquarters on NASA TV, accessible on cable networks and by classrooms across the country. Live from ExtremoFiles. We host public lectures regularly at our all research sites through “Open Night” type events. We also participate in other public events, such as panel discussions, lectures Confidential Page 75 2/12/2016 Figure A. Sample layout for the SkyWatch website, our working model and adaptable for the ExtremoFile Notebook , see hubblesite.org/skywatch. The site contains links to relevant topics, RSS feed, links to media and researcher biographical profiles and serves as a template for our Notebook portion of the website. at various institutions around our local areas (such as Maryland Science Museum, Air and Space Museum, etc.) and are regular contributors on National Public Radio such as WYPR’s Marc Steiner Show and NPR’s Science Friday. C. Christian is also the co-host of WYPR’s SkyWatch an astronomy radio vignette aired once a week and available through RSS feeds and podcasting (Figure 8.1, above), and will highlight our research advances in that venue as well. We will take all those opportunities bring the flavor of Astrobiology to the public. We intend to capture those events in our webcast archives, so that local events can reach a much wider audience. We will link those resources to the interfaces of the NAI such as the “Feature Stories” and the “Spotlight” section. 8.3 The ExtremoFile Explorer Visualizing Solar Neighborhood Data and Models. The data available in astronomical archives, accessible through the NVO, the Habitability Census, and models produced in the course of the research described in this proposal form the core of our activities in the Public Understanding of Science. Using our scientific modeling tools and observational data as a catalyst, we will create two-dimensional imagery and a three-dimensional scientific visualizations of our model solar neighborhood, down to the microbial level, collaborating with the OPO Visualization Lab. In addition, we will couple our data with existing 3-D tools available with simple viewers and browsers as well as stereoscopic goggles when possible. We make a distinction here between visualization, described above as produced by experienced animators, and the 3-D tool we will make available through a web browser. Both portray our data and models. In the one case, visualizations can be used in a variety of contexts including informal science venues, as well as classrooms. The 3-D web tool is provided by Digital Universe (DU) from ManyOne Networks, a collaborator with the NVO. The free browser tool allows the user to roam through the universe and select different scales for viewing. We will be “stewards” of the astrobiology content in the DU for the solar neighborhood, in that we will be able to add, as our research progresses, details Confidential Page 76 2/12/2016 of planetary systems and the possible life forms they can sustain, directly as links in the tool. This model can be thought of as a visually enhanced sort of Wikipedia, with robust and reliable content and authorship. Users will roam through the universe at will, and when virtually traveling through the solar neighborhood, will be able to link to supplemental information. Therefore, ManyOne Networks provides the tool, and we synergistically add value to the tool with information derived from our research results. We also intend to provide our data to the Planet Quest developers at JPL to include in their 3-D planetary systems tool, to ensure widespread discussion. The Digital Universe tool allows users to browse freely through the 3-D universe, while Planet Quest focuses on a sun-centric region, hence the two are complementary. Museum and Planetarium Displays. The visualizations produced from our scientific work will be offered as support materials to museums and planetariums along with the 2-D imagery. The materials can be integrated into projections for use by museums such as Baltimore’s Davis Planetarium, Alder and the Rose Center at the American Museum of Natural History. There, visitors will experience virtual space flight through the solar neighborhood, guided by the educator at the planetarium, describing the nature of galaxies, stars, and the astrophysical processes depicted. We will offer materials along with the other informal science modules through hubblesource.stsci.edu. As is our practice, we will coordinate with representative planetarium staff and other educators to insure resources are available in a wide variety of formats for distribution to other organizations, for example, under the auspices of the International Planetarium Society. Again with OPO, we also will produce modules for ViewSpace, OPO’s highly acclaimed multimedia product, tuned for museum kiosks, plasma screen displays, video booths, and planetarium shows and distributed widely by OPO to informal science centers. We will collaborate with OPO to provide content for STScI’s regular distribution of visualizations, video, imagery and graphical content to planetariums, documentary producers, textbook authors, and more through hubblesource.stsci.edu. ViewSpace modules comprise high-resolution images, digital movies, animation, and interpretive captions with evocative space music as a background, with content produced by a variety of astronomical facilities. The ViewSpace modules play on normal monitors, projectors, and also on high-resolution wide screen plasma displays. ViewSpace is available at about a hundred museums around the country, and OPO regularly provides updates to the program. ExtremoFile Explorer Website. Our Explorer aspect of the website will encourage the public to ask basic questions about the abundance, distribution, and types of life that may exist within our nearby neighborhood. Users will address questions through carefully crafted form-based interfaces developed as straightforward interfaces into the ExtremoFile Laboratory. Users will be able to select combinations of environmental parameters to discover the planetary systems that can host various configurations and the nature of the microbial life that can survive on planets and other host surfaces. We realize that to be successful, the ExtremoFile Explorer element of our website needs to be augmented with material that provides a useful context appropriate for the public audience. We will craft our most significant threads of inquiry to guide thinking by non-experts and motivate further reflection. Our goal is develop a public appreciation of the status of our knowledge on this subject as well as gaps in our knowledge. We will encourage the Confidential Page 77 2/12/2016 public to appreciate the types of experiments and observational data that must be acquired, and gain an additional insight into the dynamics and discovery nature of our investigations. We will create a linkage from our project to the National Virtual Observatory (NVO). The NVO EPO program collaborates with existing projects to bring real scientific data into the classroom and science centers. We will insure that our astrobiology project will coordinate relevant materials with NVO interfaces and distribution mechanisms to insure national and international access to our findings. 8.4 The ExtremoFile Laboratory We intend to share the planning, progress, and evolving results of our project with the public through cogent, understandable content based on our unique research, and appropriate for public audiences. The principal foci of our EPO activities and resources are informal science education, public information and public access as described in the previous sections, which complement the student and teacher activities already extant in the NAI suite. Our formal education support primarily will be links to existing, working, proven, curricula supplements such as Amazing Space from STScI, the COMB Microbes for Hire, Celestia, and other resources aimed at enhancing science literacy. We wish to link to specific resources available through the NAI such as Microbial Life Education Resources, Alien Safari and others. Our resources will further highlight our data, models, and the derived results so the viewer (individual, student, educator) will be able to appreciate the nature of our work in a general astronomical context. In the longer term and through the NVO EPO collaboration, we can provide data and information to our partners, such as CLEA’s2 Virtual Education Observatory where students virtually obtain astronomical data, Project Lite3, from Boston University which uses spectroscopy to explore the nature of stars, galaxies, and nebulae and the Adler planetarium’s professional development classroom4. 8.5 ExtremoFile Educators In keeping with our EPO philosophy to take advantage of collaboration with existing projects, we will partner with the UMBI BioScience Education Center and Maryland Sea Grant Extension program to develop lab-based student experiences, teacher professional development opportunities, and leverage our current teacher research fellowship programs to improve the understanding of the field of astrobiology. We will encourage studies of extremophiles biology and their potential benefit for society, and help students, through their teachers, appreciate how extremophiles could live in other habitable environments in the solar neighborhood. We will capitalize on the vibrant research in the mid-Atlantic region on extremophiles and the expertise of the University of Maryland in teacher development, and enhance teachers’ practical experience in interpreting research information on this subject. We recognize that teachers are not confident in performing laboratory activities related to extremophiles. Table 8.2, derived from the science section of the proposal, illustrates the variety opportunities of educational focus and the relation to astrophysical environments. 2 http://www.gettysburg.edu/academics/physics/clea/CLEAhome.html http://lite.bu.edu/ 4 http://www.adlerplanetarium.org/cyberspace/nvo/ - class_prog 3 Confidential Page 78 2/12/2016 With COMB in the leadership role, we will develop hands-on lab-based experiences (described below) as professional development for teachers that emphasize the value of studying extremophiles and their unique characteristic role in astrophysical contexts. Through programs that are initiated with teacher research fellowships at COMB and STScI, scientists and teachers will use the best resources currently available from our home institutions and the NAI to insure that astrobiology and extremophile research finds a path to the classrooms in the mid-Atlantic region, tied to educational standards in science, math and technical literacy. We intend to share our lessons learned and resources through exchange of materials and information to enhance our program and the entire suite of existing teacher and student programs in the NAI. Table 8.2. Extremophile Characterization. Environment Moderate: Classic habitable zone Heat: inner planetary orbits Extreme heat: close proximity to host star Cold : more typical planetary environments Acidic: early terrestrial biosphere, e.g. Salty: oceans, pods Alkaline: e.g., host springs Radiation: typical early environments Toxic: typical early environments Pressure: planetary interiors Drought: typical, e.g., Mars Rock dwelling: terrestrial planets Near starvation: probably typical Environmental Limits T~ 20 - 50 oC T ~ 50 – 75 oC T ~ 80 - 105 oC, Tmax~90 - 115 oC o C for given species T ~ -15 or below to 20 oC pH at or below 3 requires at least 0.2M salt (up to 5.2 M) pH > 10 -2 UV substance dependent tolerance up to 1.1 tons cm-3 low water activity: aw < 0.96 resident in rock low nutrients Species Mesophiles Thermophiles Extremophiles Hyperthermophile Psychrophile Acidophile Halophile Alkaliphile Radiation tolerant Toxitolerant Barophile Xerophile Endolith Oligotroph 8.5.1 Teacher Research Fellowships We will offer teachers the opportunity to become a research fellow through two established teacher research fellowship programs; the Chesapeake Teacher Research Fellowship (www.esep.umces.edu) and the VIP Expert Program (www.scienceinquiry.org) made possible with support from the NOAA Chesapeake Bay Office, the NSF and the National Park Service. Teachers will be immersed in research with COMB and STScI scientists focused on extremophile and astronomical research and ultimately apply practical ideas in the classroom setting with students. They will share ideas through the NAI as exemplary resources for their peers. We will collaborate with the teacher fellows to insure they have opportunities to make significant contributions toward professional development resources that will cascade to the Living in the Extreme Workshops. Fellowship teachers are invited to return to the workshop series in years 3-5 in order to assist in the “hands-on” components of those workshops. COMB CoIs have participated in one or both of the Fellowship programs over the last 5 years. 8.5.2 ExtremoFile Teacher Professional Development Living in the Extreme professional development workshops will emphasize the fundamental understanding of extremophile biology through interactions with STScI, COMB, and Princeton scientists. These workshops will offer the opportunity to perform “hands-on” lab experiences developed for safe use in the classroom by education and outreach staff at The UMBI Confidential Page 79 2/12/2016 BioScience Education Center in partnership with MD Sea Grant. We intend that this activity have synergy with the NAI Summer Teacher workshop, although as of this writing the content of the latter workshop is not yet available. We base the Living in the Extreme professional development workshop upon a previous workshop, Microbes for Hire, sponsored by the Pfizer Foundation, The Foundation for Microbiology, and Bell Atlantic-Maryland held in 1998, 1999, 2000 and 2003 at COMB. The workshop hosted Maryland middle school and high school teachers and was designed to translate the applied microbial ecology research at COMB into laboratory activities for teachers and students in Maryland. In Microbes, teachers gained laboratory skills and enhanced content knowledge on a variety of topics including bioluminescence, bioremediation and the Winogradsky marine sediment colonial growth stimulation effect. Similarly, Living in the Extreme teachers will gain experience with biological activity in relation to surrounding environments, and be able to relate this knowledge to astrophysical contexts. Each topic area will be formulated in collaboration with COMB and STScI scientists or graduate students to provide the essential background of the research and followed by a "hands-on" labs that expose teachers to new research techniques, and ideas for their classrooms. We will also use the Living in the Extreme workshop as an opportunity to investigate cultivation of extremophiles in science museums. We will partner with the Exploratorium in San Francisco, an innovative museum that connects science with art and human perception. The Exploratorium’s hands-on science museum is visited by half a million people each year; their website draws roughly 20 million different visitors per year. They have an active program for training teachers to develop creative science curricula, and they also design and produce exhibits for other science museums around the world. The Exploratorium recently launched the “Microscope Imaging Station” (www.exploratorium.edu/imaging_station/about.html) that puts museum visitors in control of research grade phase contrast and fluorescent microscopes to study cultures of mouse stem cells, embryonic heart development in zebrafish and round worms. Two members of the Exploratorium exhibits staff with formal biology training will attend the Living in the Extreme Workshop. They will learn techniques for cultivating various extreme microbes and the planetary environments in which they might be found. In follow-up teleconferences and videoconferences, proposal team members will collaborate with Exploratorium staff on the establishment of continuous cultures of model organisms in the Exploratorium’s laboratory facilities and follow up with in situ visits to devise a plan for the establishment of extremophile cultures and supporting astronomical content for an exhibit, related to the Microscope Imaging Station. This work will build on the success of an NAIsponsored collaboration between UMBI and the Exploratorium on documenting extremophile research in Uzon Caldera, Kamchatka, Russia, in the summer of 2005 (NAI Director’s Discretionary Fund award to UMBI Collaborator Albert Colman). Exploratorium also has a long history of collaboration with STScI for astronomical exhibits and a webcast series based on servicing of HST. Confidential Page 80 2/12/2016 8.6 Evaluation Formative evaluation of our EPO program will be directed by an evaluator from OPO at STScI. The evaluation criteria will be integrated at the initiation of the program. Assessment of the program will be effected through a number of activities including: observation of teacher workshop participants during the preparation phase and follow-up with participants afterwards. We envision interacting with typical web users at various meetings such as the National Science Teachers Association and Association of Science and Technology Centers to probe the usability and effectiveness of our resources in sit-down sessions in display booths at those meetings. We wish to probe the preconceptions held regarding astronomical research on planetary systems and extremophile research and the linkages between them. We especially intend to be attentive to how our efforts are unfolding and uncover obstacles, barriers or unexpected opportunities that may have emerged. For summative evaluation we will work with informal and formal science educators who use our resources and collaborate in our workshops. We will evaluate the extent to which our resources stimulate usage of our astrobiology material in various forms of education. We will also take advantage of built in measures of web sessions and threads to understand how each section of the web resources is being used and the linkages users make between various areas. 8.7 Our Timeline We will use a staged approach for our Education and Outreach program. We consider that in our first year, we will be creating the basis of our project so that production of fully developed educational materials would be premature. We briefly describe the timeline here: Year 1: The EPO Lead (Christian) will become integrated into the overall NAI education and outreach network, developing an understanding of what products and services are available and in development and will share the team's plans for the future. The Lead will coordinate support of other NAI EPO activities and provide unique resources as appropriate. We will initiate the Live from the ExtremoFiles public lectures program, radio programs, and webcast archive. Initial public information on especially the Notebook part of the website will be developed. We will develop the initial evaluation program and metrics plan. We will also begin the teacher fellowship programs to partner 4 teachers with COMB and STScI Scientists. We will populate the Digital Universe with existing baseline habitability data. Year 2: The EPO Coordinator/Lead will evolve the detailed education and outreach plan based on current needs assessments of the overall NAI and NASA programs and the intended audiences. The EPO team will develop public access requirements for science derived products and initiate development of the ExtremoFile Explorer part of the website. The Notebook will continue to carry the latest information and news on the project. Teacher fellowships and professional development workshop planning will continue. Year 3: We will augment the public portal with enhancements to the ExtremoFile Explorer. The 2-D and 3-D visualizations will be initiated, based on preliminary data from the Habitability Census. We will augment the Digital Universe each year as new data and information becomes available. Evaluation on the website and the workshops proceeds. The Teacher Fellowship will end at the conclusion of year three and planning and recruiting teachers from the Living in the Confidential Page 81 2/12/2016 Extreme professional development workshop will continue. The Notebook will be maintained. Year 4 and Year 5: We will develop and disseminate planetarium and museum renderings of science data visualizations. The team will create ViewSpace feature programs, other materials for educational use and continue to make press announcements as appropriate. We will create links with NVO during the last year of the project. Teacher fellows from years 1-3 will return to assist with the implementation of the one-week Living in the Extreme professional development workshops held at the COMB during the summer months of year 4-5. Enhancements to the Digital Universe and the Notebook continue regularly. In the last year, the final evaluation report will be generated. 8.8 Our Education Expertise STScI, COMB and Princeton have histories and extensive experience in developing various curriculum support materials for education. Our experience also extends to support of pre-service and in-service teacher education at a variety of colleges and other educational institutions across the country. OPO at STScI has established itself as a major leader in providing resources based on NASA space science that are directly relevant to mathematics and science curricula. OPO produces a complete suite (Table A) of educational and outreach materials from news materials and support of media, to the public information site (hubblesite.org), to the formal education support under the umbrella of Amazing Space, and informal science education resources. At COMB, the Microbes for Hire Program is designed to give current and prospective middle and high school teachers within Maryland the skills, experience and confidence that they need to effectively integrate information and imaging technology with the science curriculum and to teach an increasingly important area of science – biotechnology. The innovative program is part of an ongoing commitment to public outreach and inquiry-based education by the University of Maryland Biotechnology Institute’s COMB and Maryland Sea Grant College. They have successfully provided hands-on experiences in marine biotechnology to over 15,000 Maryland students and their teachers over the past few years in the Science and Technology (SciTech) Education Program Labs located in the Columbus Center, Baltimore, MD. Teacher Research Fellowship programs (VIP ExPERT and Chesapeake Teacher Research fellowships) are well established and illustrate the value of the teacher-scientists relationship. These programs also take advantage of the unique opportunity to establish a direct pipeline between research scientists and classroom teachers that will continue beyond the period of the grant once there relationships are formed. Confidential Page 82 2/12/2016 9. References 9.1 Research and management Adams, F. C. & Spergel, D. 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Appendix: Glossary of terms and acronyms 2MASS AAS ACS ACP AMASE AO APO AU CCD CIDS CIW COEL COMB CPD CSD CSP CTIO DMSO DNA DOE EATT E/PO ESA ESO ExSUF FISH Gaia Gemini GMT GSFC Gyr HiCIAO Hipparcos HST IAC JGI JWST KPNO LINAC MARTE MAST Myr Confidential 2-micron All-Sky Survey, near-infrared sky survey American Astronomical Society Advanced Camera for Surveys (a CCD imaging camera on HST) Astrobiology Certification program (at PU) Arctic Mars Analog Svalbard Expedition Adaptive Optics (astronomical technique for image sharpening) Apache Point Observatory Astronomical Unit, the Earth-Sun distance Charge-coupled device Circular intensity differential scattering Carnegie Institution of Washington Committee on the Origin and Evolution of Life (National Academy of Sciences) Center of Marine Biotechnology cyclobutane pyrimidine dimers cold shock domain cold shock proteins Cerro-Tololo Interamerican Observatory, Chile Dimethyl sulphoxide Deoxyribonucleic acid Department of Energy Extremophiles in Astrobiology: Theory and Techniques Education/Public Outreach European Space Agency European Southern Observatory Extreomphile scale-up facility Fluorescence in situ hybridization ESA astrometric mission, scheduled for launch in 2015 US 8-metre class telescopes on Mauna Kea, Hawaii, and Cerro Pachon, Chile Greenwich Mean Time Goddard Space Flight Center Gigayear High Contrast Coronagraphic Imager with Adaptive Optics ESA satellite that measured accurate positions, motions and distances for 118,000 stars, including many in the Solar Neighborhood The Hubble Space Telescope Idaho Acceleration Center, Idaho State University Joint Genome Institute The James Webb Space Telescope Kitt Peak National Observatory Linear accelerator at the IAC Mars Analog Research and Technology Experiment Multimission Archive at STScI: an archive of data from several space missions Megayear Page 97 2/12/2016 NAI NAOJ NASA NIC-FIPS NIST NOAA NSF NVO OGLE OPO ORF NASA Astrobiology Institute National Astronomical Observatory of Japan National Aeronatics and Space Administration Near-infrared camera – Fabry-Perot Spectrograph (at APO) National Institute of Standards and Technology National Oceanic and Atmospheric Administration National Science Foundation National Virtual Observatory Optical Gravitational LEns survey Office of Puiblic Outreach, STScI Open Reading Frame, the DNA or RNA sequence between the start and stop codon sequences parsec Astronomical distance unit, corresponding to 3.26 light years or 206265 AU PCR Polymerase chain reaction PEM Photo-elastic modulator PEMS Portable Earthsine Monitoring System PU Princeton University RNA Ribonucleic acid rRNA recombinant RNA RSS really simple syndicate SAO Smithsonian Astrophysical Observatory SIMBAD An astronomical database maintained at the University of Strasbourg SIM Planetquest The Space Interferometry Mission, a NASA mission designed to obtain high precision positions and detect planetary companions to nearby stars SMARTS Consortium of universities and research institutions that run the smaller telescopes at CTIO SNH Census Solar Neighborhood Habitability Census STIS Space Telescope Imaging Spectrograph STScI Space Telescope Science Institute TMAO Trimethylamine N-oxide TPF The Terrestrial Planet Finder TPF-C The Terrestrial Planet Finder: Coronagraph TPF-I The Terrestrial Planet Finder: Interferometer Tycho Auxiliary star catalogue produced by the Hipparcos satellite UCLA University of California at Los Angeles UMBI University of Maryland Biotechnology Institute UNSW University of New South Wales VLT Very Large Telescope (Four 8-metre telescopes at the ESO Paranal site, Chile) VPL Virtual Planetary Laboratory (http://vpl.ipac.caltech.edu/) VRML Virtual reality markup language Confidential Page 98 2/12/2016