DRAFT
Life in Extreme Environments
(LExEn) Workshop Report
Workshop held:
June 17-19, 1998
at the
National Science Foundation
4201 Wilson Boulevard.
Arlington, Virginia 22230
Steering Committee:
Jean Brenchley, Penn State University
David Karl, University of Hawaii
Julius Jackson, Michigan State University
John Delaney, University of Washington
Jack Welch, University of California, Berkeley
Table of Contents
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Page
I.
Introduction.................................................................................................................
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0
What conditions determine the limits of life?........................................
4
Introduction.................................................................................................................................
Specific Research Topics........................................................................................................
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5
5
1.Energy....................................................................................................
2.
Tolerances.............................................................................................
3. Cell structure..........................................................................................
4.
Resources................................................................................................
5. Possible foci for future
research............................................................
Methods, Technology, and Infrastructure..............................................................
1. Methods development............................................................................
2. Technology enhancements.....................................................................
3. Infrastructure support............................................................................
4. Community building...............................................................................
III.
What is the functional diversity of
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life?...................................................
Introduction............................................................................................................
Specific Research Topics.......................................................................................
1. Physiology and structure........................................................................
2. Group and species interactions..............................................................
3. Stress responses......................................................................................
4. Dispersal.................................................................................................
5. Indicators of biological processes..........................................................
6. Microbial diversity data.........................................................................
7. Evolutionary adaptation to extreme environments.................................
8. Possible foci for future
research............................................................
Methods, Technology, and Infrastructure...............................................................
1. Methodology...........................................................................................
2. Community Building...............................................................................
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IV.
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How does life evolve from pre-biotic
conditions?...................................
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Premises..................................................................................................................
Specific Research Topics........................................................................................
1. Did life on earth originate in an ‘extreme environment’?......................
2. What is the range of extreme environments in which life can................
originate?
3. Where did environments conductive to the origin of life exist?.............
4. What were the conditions under which life evolved?.............................
5. How do fluctuations in physical and chemical environments affect the
evolution of organisms?
6. Are there unique genetic features of life in extreme environments?......
7. Have extreme environments been central to the evolution of life?........
Methods, Technology and Infrastructure................................................................
1. Encourage theoretical development.......................................................
2. Technology development........................................................................
3. Community building...............................................................................
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V. Appendices
A. LExEn Program Announcement FY98..............................................................
B. LExEn Workshop (June 17-19, 1998) Agenda..................................................
C. LExEn Workshop Participants...........................................................................
D. Some LExEn Questions.....................................................................................
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I. Introduction
The research activity called Life in Extreme Environments (LExEn) was formally
announced by the National Science Foundation on January 21, 1997 to facilitate
coordination of the study of "extreme environments" and the often abundant, microbial
life found to flourish in them (Appendix A). The LExEn Activity resulted from the
combined intellectual and funding participation by the Directorates for Biological
Sciences (BIO), Geosciences (GEO), Mathematical and Physical Sciences (MPS),
Engineering (ENG), and the Office of Polar Programs (OPP) at the NSF. In general, the
initial aim of LExEn was to enhance knowledge about life in extreme environments
through highly interdisciplinary, integrated research programs exploring the relationships
between organisms and the environments within which they exist. A strong emphasis
was placed upon those life-supporting environments that exist near the extremes of
planetary conditions. An important component of the study of life-supporting
environments was to assist in the exploration of planetary environments in our solar
system and beyond and identify possible sites and conditions that may have supported or
still supports life elsewhere. The LExEn Activity was launched with a commitment to
focus resources to enhance the understanding of the microbial systems on Earth,
particularly with respect to their diversity and the mechanisms that allow microbes to
survive and alter extreme environments. The initial phase of this LExEn Activity was
expected to catalyze the collaboration of scientists from multiple disciplines; to
encourage the development of instrumentation and technologies that would enable remote
sampling and sensing; to expand discovery about the diversity, ecology and physiological
capabilities of microbes; and to increase knowledge about the geological and evolutionary
history of microbes that inhabit the extreme environments on Earth.
NSF convened a LExEn workshop in June of 1998 to develop recommendations
for future directions of the LExEn Research Activity. The agenda for the workshop and
the list of participants can be found in Appendices B and C, respectively. The
underlying principle sustaining this Activity is that the achievements should be greater
than what could be attained by the routine award of single, investigator-initiated research
grants. The organizing strategy for this Workshop was to form three Primary Questions
for the participants to address, and to look at these questions through a LExEn lens. The
Steering Committee for the LExEn Workshop recommended a preliminary set of Primary
Questions to start the discussions, and all Workshop participants engaged in developing
and defining the final set of questions for working group action.
Primary Questions
1. What conditions determine the limits of life?
2. What is the functional diversity of life?
3. How does life evolve from pre-biotic conditions?
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Working Group Charge
Working groups were organized to address each Primary Question and each group
was given two broad charges.
 Develop an "Investigative Strategy" directed at answering the Primary Question.
 Develop ideas about how to build a LExEn community of scientists, i.e. how to
nurture and develop the interdisciplinary research teams necessary to make
progress with LExEn objectives.
The Investigative Strategy was defined as a hierarchy of questions beginning with a
single Primary Question and ending with a multitude of specific questions of sufficiently
limited scope that they might be undertaken within a single, conventional research
proposal. Between these two extremes is some undefined number of levels of questions
of ever-increasing specificity. The working groups were asked to think of and develop
significant questions that may be answered by long-term (a human lifetime), mid-term (a
scientific career), and near-term (1-3 regular funding cycles) investigative strategies.
The workshop participants were asked to develop questions strictly based upon the
science of LExEn without regard to considerations of resource requirements.
The following report consists of summaries of the three working groups, each
assigned to one of the Primary Questions. In their summaries, they present Specific
Research Topics that include a wide range of general as well as specific questions to be
answered through basic research. They also present Methodologies, Technologies, and
Infrastructures that cover instruments and methods necessary for research in extreme
environments, as well as community networking to encourage interdisciplinary action.
Considerations and Expectations
Working groups were asked to define the significance of the Primary Question
being considered and construct an overview of a hierarchical structure for the
representative questions developed for each time scale. At each level of the hierarchy
the discussions were to produce representative examples of the most significant questions
to be answered in order to gain new scientific understanding and insight.
Several specific points of guidance for the working groups were agreed upon at
the outset:
 In choosing the representative questions, consideration should be given to the
maintenance of an appropriate balance between the study of life forms and the
study of the environments within which they exist.
 Where possible, questions should be posed in such a way as to optimize the
potential benefit of interactions between disciplines.
 The importance of exploration and discovery-oriented research approaches should
be recognized as a fundamental emphasis in addition to the more traditionally
favored emphasis upon hypothesis testing.
 As appropriate, brief descriptions should be provided of the infrastructure,
capabilities and technology required to achieve the stated research objectives.
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A set of specific LExEn questions compiled by Hugh Van Horn following the initial
workshop presentation was also provided to the working groups, and is included as
Appendix D.
The expected outcome of the 1998 LExEn Workshop is new insights into the
science that is LExEn, through a set of recommendations to establish ambitious and
achievable goals that will guide this area of research well into the 21st century.
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II. What Conditions Determine the Limits of Life?
Introduction
A large population of organic molecules has been discovered in the interstellar gas
with radiotelescopes. Space craft have recovered evidence that water was once plentiful
on the surface of Mars and that the Jovian satellite Europa may be covered with ice, and
there is the possibility that a fairly deep ocean lies beneath. Finally, over the last two
years Jovian scale planets have been detected around at least a dozen nearby stars, with
many more such discoveries probably soon to come. The astronomical setting for the
origin of life is a scientific discipline likely to develop explosively over the coming
decade.
On planet Earth, there is a recognition that contemporary environmental conditions
are not the norm, and that habitats more hostile to life as we know it probably existed in
the past. The geologic and fossil record offers an opportunity to gain insights into the
history of extreme environments on Earth and may offer a perspective on the potential for
life on other planets.
Life has successfully radiated into many diverse environments on Earth, including
those that might be termed “extreme.” This designation is anthropocentric and many
extremophiles (organisms inhabiting unusual environments) cannot compete successfully,
or even survive outside of these extreme habitats. An understanding of life processes in
extreme environments will allow constraints to be placed on the limits of life.
A working definition of an extreme environment is one where physical and chemical
conditions approach or exceed the tolerances for life. However, it should be emphasized
that some organisms actually thrive even under selected extreme environmental
conditions. Common examples include high temperature fumeroles and high salt content
evaporite deposits. More subtle extreme environmental conditions may also include
habitats that contain only dilute concentrations of life sustaining nutrients or those with
unusually high concentrations of the same solutes. Many of these unusual environments
are extreme by more than one criterion. For example, the deep-sea is a high pressure,
low temperature habitat, and some hypersaline lakes in Antarctica have high salt
concentrations and very low temperature. Many extreme habitats are inaccessible and
have not been adequately characterized or even explored. A key requirement of any new
program of life in extreme environments is the need to integrate life processes with a
detailed study of the environmental conditions.
There are both intellectual and practical reasons for undertaking ecological studies of
extreme environments. First and foremost relates to the origin of life itself. While even
a comprehensive study of life in extreme environments will not necessarily provide the
answer, scientific investigations in the full range of habitats in our universe will provide a
better understanding of how our planet functions from geological through biological
processes. These additional insights will provide much needed data on chemical fluxes
and climate variability as well as the biological consequences of climate variability.
There are also many potential practical applications of this information from
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biologically-mediated materials production, to natural resource preservation, to “biochip”
manufacturing and other biotechnological applications.
Specific Research Topics
1. Energy
Life that is familiar uses either light or chemical energy. Are there other forms of
energy (e.g., thermal, radiative, magnetic) that life can access directly for growth and/or
maintenance? It may be possible to answer this question by examining it in terms of life
in extreme environments:
 What is the minimum amount of energy required for growth and/or maintenance?
 How is energy extracted from the environment?
 How efficient is the extraction of energy?
 What is the minimum life-supporting energy supplied by an environment?
One goal of the LExEn program is to identify environments that can support life
either on other planets or in less accessible regions of the Earth. What can the study of
extreme environments reveal about novel life-supporting conditions?


What is the full diversity of electron and carbon sources used by life?
What are the requirements for environmental/energy continuity?
Taking advantage of novel energy sources may require novel metabolic strategies:

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

What is the full diversity of metabolic strategies used by organisms, and what is
the range within an individual organism?
Are there alternates to ATP as methods of intracellular energy transfer
systems?
What is the energetic demand for acquiring rare (dilute) resources?
What is the fate of marine dissolved organic matter that enters hydrothermal
systems? Can it be converted to bioavailable substrates?
2. Tolerances
The distribution of life on Earth occurs within bounded ranges of chemical and
physical parameters. Such chemical parameters may include water activity, redox
conditions, pH, salt, nutrients, trace elements and other solutes including toxic elements
and compounds.
The physical aspects of environments are parameterized by
temperature, pressure, level of ionizing and non-ionizing radiation and are described as
solid, liquid or gaseous and in a tectonic context. Thus these physical and chemical
parameters also have a temporal dependence ranging from the time scales of chemical
reactions to billions of years. The importance of high and low frequency habitat
variability and stochastic events (impacts, volcanoes) may be very important
considerations.
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The tolerances and adaptability of organisms within combinations of these parameters
establish the limits to the dispersal and distribution of life on Earth and may help both to
infer the existence of life on other planets and the exchange of life among planets (via
meteorite transport, for example.)
The problem of the limits of life within these parameters is a multifactorial one.
Among the studies which will elucidate the limits of life include the following:

Determining the distribution of organisms, including dormant forms in extreme
environments

Assessing where life on Earth exists in a functional state

Examining how organisms respond when placed at sets of parameters that are
close to limiting values

Studying the extent that the existence of life at limiting conditions facilitated by
community structure

Analyzing the fossil history of life on Earth to find out how changes in Earth’s
physical and chemical environment have influenced the limits of life over time;
using the fossil and geological record to examine tolerance limits

Determining the geochemical composition of habitats and of planetary
environments that are candidates for harboring life

Examining the dynamical evolution of planets in the context of the generation of
conditions which are habitable

Studying how organisms and chemical resources could survive entry into Earth’s
atmosphere.
3. Cell structure
Contemporary cells have evolved certain features to ensure their existence in a wide
variety of niches on Earth. Some of these features are common to all or most cells;
others are unique for particular physiologies and conditions. One limit for life, crudely
defined here as the ability to reproduce and perform regulated metabolic processes, are
the minimum essential cellular components. In the context of LExEn several questions
are particularly relevant. These include:
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What is the minimum cell size? How do extreme conditions influence size?
How is this related to the nutrient status of the environment?
What is the historical record of cell size and does it suggest adaptations to
extremes?
Do organisms develop protective structural adaptations to extremes? For
example, do cell walls, membranes, shapes and communities (symbiotic
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structures) adapt to armor cells so that they can live at the limits of life? What
might be reasonable postulated strategies not yet seen?
Are there strategies for enzyme-substrate channeling that may enhance adaptation
such as retention of resources, production of metabolic water, avoidance of
inhibition?
What is the role of Eukaryotes in the generally Prokaryote-dominated extreme
environments?
4. Resources (other than primary energy requirements)
Microorganisms possess qualitative and quantitative requirements for diverse
resources that are essential to support life. Requirements vary according to species,
metabolic state and micro-environmental conditions. Water, C, N and P are presumed to
be basic to all life, although required levels vary. Other resources, such as some enzyme
trace metal requirements, can be organism-specific and substitutions are possible. In
considering resource requirements, it is critical to explicitly distinguish between resource
classes and levels that are required for growth and those required for survival alone.
Progress in research related to the “resource” questions will benefit greatly from
environment-specific geochemical models. Examples of questions related to life’s
resource requirements include:

What are the obligate requirements for major and minor elements?
– What are the acceptable physical-chemical species for these
resources?

What is the minimum water activity?
– Is liquid water required?
– Can water vapor or chemically bound water (e.g., waters of hydration) satisfy
water requirements?

How are primary resources supplied to organisms, especially under resource
limitation conditions (e.g., comets vs. planetesimals/ fluid flow / chemical
transport)?
–
–
–
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What resource acquisition strategies are employed by organisms, especially in
resource limited conditions? (e.g., dissolution of solid substrates)
What is the minimum rate of supply of limited resources? How are they
delivered?
What are the geochemical sources/processes that provide key resources?
How quickly can microbes adapt to changing inputs in key resources?
– What mechanisms enable microbes to respond to changing resources?
– How are resource sources/acquisition strategies affected by trophic
interactions?
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What potential chemical resources are present in extraterrestrial material (comets,
asteroids, interstellar dust)?
5. Possible Foci for Future Research

Exploration and characterization of key habitats through coordinated
interdisciplinary field studies for gaining a functional understanding of past and
present extreme environments. This provides insight into what is novel and tells
how system functions.

Laboratory studies based on realistic ecosystem models designed to elucidate the
mechanisms of microbial growth and survival.
Methods, Technology, and Infrastructure
The continued search for the outposts of life will demand several technology
enhancements as well as the development of novel field sampling and analysis procedures
to reach our stated goals. There are also key infrastructure and other support facilities
that need to be dedicated to these programs.
The ability to monitor extreme environments and the response of these environments
and of their inhabitants to natural perturbations would be a valuable outcome of the
development of in situ measurement and sampling techniques. Sampling of material
present in space and improved (higher spatial resolution) detection and characterization of
planetary environments are also desired goals of the LExEn program that can be
addressed in concert with other government sponsored programs.
Investigation of the factors that limit life will also require laboratory studies carried
out in appropriate conditions. Instrumentation for thermodynamic and kinetic studies
and for laboratory spectroscopic studies of reactive molecules will need to be upgraded.
Technology required for reproduction of extreme environments in the laboratory, and for
studies of extremophile physiology, will need to be developed. Modeling efforts also
need to be enhanced.
1. Methods development
Improvements and innovations are also needed in methods used for in situ sampling
in extreme environments to allow characterization of habitats and inhabitants. This
includes development of:
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Methods for preventing contamination of samples and methods for measuring key
variables in situ
Methods for in situ identification of the presence of microorganisms and for
sampling micro-environments
Proxies (e.g., isotopic, biomarkers, trace element) for specific metabolic activities
or microbial assemblages
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Novel methods for primary isolation, purification and growth of extremophiles
Novel, sensitive methods to measure rates of microbial activity and probes for in
situ activity
Cellular, geochemical and thermodynamic modeling capabilities to assimilate new
knowledge and to guide future research efforts

2. Technology enhancements
A major goal of LExEn will be sampling of the full range of extreme environments,
requiring improvements in current methods of obtaining in situ samples. Examples
include:

Enhancement of Ocean Drilling Program (ODP) drilling technology to allow:
prevention of contamination of drill-core
retention of volatiles
recovery of cores from extreme ocean depths
recovery of material from directly beneath active vent sites
Installation of a microbiological laboratory on board the ODP drilling vessel
Development and enhancement of drilling and coring techniques for ice and
terrestrial habitats
Adaptation of existing CORK (Circulation Obviation Retrofit Kit) technology for
subsurface microbiological studies
Alternative methods of obtaining samples from extreme environments, such as use
of smaller sea-floor drilling platforms
Sampling of event plumes and of the range of hydrothermal effluents exiting the
seafloor as a means of sampling sub-surface environments
Development of platforms for aerobiology
Planetary sample return and analysis
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3. Infrastructure support
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Information storage and retrieval (e.g., ribosome database, and key habitat and
phenotypic data)
Develop and maintain extremophile culture collections
4. Community building
Many new insights into understanding life at its extremes are likely to come from
sharing of knowledge amongst disciplines that have not traditionally interacted, e.g.
microbiology, planetary science and astronomy, geology, geochemistry, paleontology,
molecular biology and engineering. To ensure multidisciplinary potential is realized we
recommend the following:


A post-doctoral program that requires training in a new relevant discipline
An interdepartmental pre-doctoral training program
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Cross-disciplinary workshops similar to RIDGE Theoretical Institute (RTI) with
representation from advanced graduate students, post-docs and scientists. The main
workshop product will be a “life in extreme environments” reference document, in
easily understood language, which articulates the cross-disciplinary program goals.
This will be distributed to the peer review community with each submitted research
proposal
Dedication of a portion of the total resource allocation to collaborative projects.
The panel that reviews these proposals must be carefully selected to ensure that it is
appropriate for this criterion
Allocate a portion of program resources to small grants to exploratory research
(SGER) grants
Develop and maintain a web page with relevant cross-disciplinary data sets,
publications and other information that could serve to stimulate collaboration.
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III. What is the Functional Diversity of Life?
Introduction
Extreme environments on Earth represent some of the best analogues for
understanding life's development and its potential existence on other planets. These
environments span diverse physical, chemical, and biological extremes that are often
harsh, remote, and very difficult to sample, observe, and study. Extreme habitats, by
their very nature, are currently not thoroughly characterized or well understood.
Understanding the functional diversity of extreme environments and their indigenous
organisms will require detailed information derived from and integrated between several
different hierarchical levels. These include functional diversity at the chemical/molecular,
biochemical, cellular, multicellular/consortial, and ecosystem levels. Understanding
extreme habitats requires knowing "what's there" in terms of habitats and organisms,
"what they're doing" in terms of function, and "how they’re doing it", in terms of specific
adaptations and properties of life in extreme habitats. Achieving these goals necessarily
requires research that hinges on the integration of knowledge between multiple
disciplines, from diverse molecular biology to radio astronomy.
Community, organismal, and molecular processes in extreme environments will be
examined in diverse ways including: bioenergetics (light, chemical energy), uptake of
substrates (carbon compounds, other nutrients), physiology (metabolic processes),
community interactions (syntrophy, symbiosis, competition, genetic exchange, controls
on levels of activity), preservable indicators of biological processes (biomarkers), and
evolutionary adaptation of metabolic processes to extreme environments.
The processes of life in extreme environments parallel those in ‘normal’
environments yet require specialized adaptations to achieve those ends. Extreme
environments impose extraordinary constraints upon the organisms that live there. This
section addresses specific mechanisms at each of the levels of functional diversity
discussed above (molecular, biochemical, cellular, multicellular/consortial, ecological
and evolutionary) that allow organisms to inhabit these environments. The reciprocal
interaction between environments and living systems (at the entire range of scales) needs
to be documented using interdisciplinary approaches.
Laboratory and field
investigations of these questions necessitate the development of new technical and
analytical methodologies due to the extreme nature of these environments. Information
gleaned from these studies will be useful for the recognition of life in other contexts (on
Earth and other planets), the identification of commercially useful products,
biotechnology and the utilization of basic technology and instrumentation developed in
this area. We need to learn the general principles involved with life in extreme
environments due to the extreme range of environments, species, and communities.
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Specific Research Topics
1. Physiology and Structure
This research is driven by the desire to understand what features extremophiles
possess which allow them to inhabit environments that are prohibitive to other life forms.

What are the unique physiological features of individual extremophiles that yield
an advantage for growth and survival in an extreme environment?

What are the shared features within a group of extremophiles (i.e.,
hyperthermophiles) from one type of extreme environment?

What are the shared features between groups of extremophiles (i.e., between
psychrophiles and barophiles)?

Do extremophiles evolve convergent mechanisms to solve a common
environmental problem?

How were these specialized physiological features acquired?

What are the energetic costs and benefits of adaptation to extreme environments?
These studies require the development of methods to detect and analyze cellular
components that are not routinely examined in biology.

Are there unique examples of the following in extremophiles: cofactors, proteins,
cytosolic solutes, membranes, electron carriers, energy currencies, metabolic
pathways, nutrient acquisition mechanisms, transcriptional and translational
mechanisms?

Are special life history strategies necessary in extreme environments?
One of the challenges encountered by microorganisms in extreme environments is
the physiological and structural adaptations required for basic physiological processes
including growth, reproduction, and cell maintenance. These questions must be
approached at several levels of cell metabolism.

What is the full range of potential energy sources, including expanded ranges
of known sources and new sources, and the bioenergetic mechanisms for
harvesting these sources? Also, what is the metabolic basis for hierarchical,
complimentary, or competitive energy utilization within and among
microorganisms?
Microorganisms also have requirements for nutrients that serve as building blocks for the
synthesis of cell monomers and polymer.

Are there nutrients used by extremophiles that have not been previously
identified as biological components and new biochemical paradigms for
assimilating nutrients into cell components?
Cell structure may also have a critical role in the specific adaptation to extreme
environments.

Are there specific structural adaptations (i.e., intracellular macromolecules,
cell envelop, etc.) that cells have developed to survive and proliferate in
extremes and during fluctuations between optimal and non-optimal
environmental conditions?
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
Are there behavioral responses to extreme environments such as attachment
mechanisms and motility (i.e., chemotaxis, thermotaxis, phototaxis)?
Research on these topics requires identification of metabolic pathways and
mechanisms of control at both the biochemical and genetic levels. Specific physiological
extremes include: high and low temperature, low water activity (desiccation, halophily),
high and low pH, high ionizing radiation, high pressure, low nutrient concentrations,
extremely low and high redox potentials, environmental fluctuations.
Identification and characterization of novel metabolism will require
multidisciplinary approaches. Examples of possible collaborations for conducting
research on microorganisms with not previously described metabolisms would include:
- chemists, geologists and mathematical modelers to predict the possibility of
growth in the context of specific extreme environmental parameters
molecular biologists, microbiologists and engineers to develop novel
approaches for isolation and sustained growth of novel microorganisms
- biochemists, molecular biologist and astronomers to identify remote and in situ
methods to detect terrestrial and extraterrestrial metabolic processes
- mathematical modelers, engineers, molecular biologists and biochemists to
determine molecular parameters for macromolecular structure/function under
extreme conditions.
1. Group and species interactions
The following topics will be discussed: the transfer of specific nutrients among
community members, the degree of specificity of interactions within a community, the
extent to which species are trophic generalists or specialists, the presence or absence of
‘keystone’ species, endosymbiosis and the degree to which the community is necessary
for the survival of individual species, and the implications of the presence or absence of
specific physiologies upon community structure.
The degree to which species interactions and community function in extreme
environments differs from that in more benign environments is not known. The
energetic costs associated with adaptation to extreme environments may limit the number
of trophic levels. Conversely, the exclusion of many larger organisms from extreme
environments may release communities from ‘top down’ control (structure in the
environment imposed by predation and grazing). Specific metabolites produced in
extreme environments may be a currency of exchange among species or serve as
alternative nutritional resources as fluctuations in the environment occur. Specific
physiological adaptations may structure interactions among species in extreme
environments. Communities may become more or less structured and functional
relations among species more or less coupled as life approaches the limits. For example,
approaches to the limits of life may require symbiotic interactions and consortia (sets of
organisms achieving ends that are attained by single organisms in other environments) to
carry out ecological functions. The degree to which ‘keystone’ species play an important
role in extreme environments is unknown. It is not clear whether the boundaries to life
in terms of physiology coincide with the ecological boundaries (can extremophiles in pure
culture survive closer to the limits of life than can a consortia or in situ ecosystem or vice
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versa?) Little is known about community ecology in any extreme environment.
Investigation of species interactions in extreme environments should elucidate general
underlying principles of ecological organization and how they are constrained by the
physico-chemical environment. Qualitative assessment of communities (who is there)
needs to have an explicit link with rigorous quantitative analysis of abundance within and
across ecosystems (at a single location, between locations and globally). The number of
species, their identities, biomass and a frequency distribution need to be documented.




Biofilm architecture – Role of cellular aggregates and exopolymers in stabilizing
the community and facilitating the harvesting of energy and the flow of chemical
species among community members. There is a need to understand the response of
this architecture as a part of the adaptation to particular environments.
Syntrophy, symbiosis, competition – Need for baseline studies of such interactions
within extreme environments as a basis for understanding their role in the adaptation
and survival in extreme environments.
Genetic exchange – Need for baseline studies of transformation, etc. processes as
factors in developing the genetic ensemble in extremophiles. For example,
delineate the roles of gene transfers in the diversification and dispersal of molecular
adaptations of extremophiles.
Controls on levels of activity – Need to delineate “who is active and inactive” in
these communities, as a basis for discriminating between modes of growth and
survival in extreme environments.
3. Stress Responses

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


What are the stress and survival mechanisms employed by extremophiles?
Are the mechanisms of an extremophile to deal with environmental stress the same
as in non-extremophiles, or do they possess novel mechanisms?
Are stress mechanisms constitutively expressed by extremophiles, or are their
cellular components specifically adapted for the environment?
How do extremophiles survive exposure to environments outside of the
environment they are adapted to?
Examples of stress responses to be examined: heat or cold shock, pressure stress,
osmotic stress, nutrient deprivation, pH and Eh variation, the availability of water,
and dormancy/encystment.
4. Dispersal
Extreme environments are frequently patchy, ephemeral and unpredictable. This
structure requires organisms to evolve novel mechanisms for persistence during times of
environmental change or for the colonization of new areas. Many organisms from
extreme environments have also been detected in a wide variety of environments outside
of their ‘typical’ habitat. The ecological tolerance of these organisms may be extremely
broad, or there may be special adaptations to allow persistence in a resting state.
14
Denizens of extreme environments appear to be highly adapted to specific sets of
conditions, but these habitats tend to be patchy, or even widely separated, and sometimes
ephemeral. Some adaptations seem to be at the ecosystem level.

How then do these extremophiles manage to find and colonize appropriate sites?

It would seem inefficient to blanket the earth with germ plasm at random, yet what
mechanisms could guide individuals or community members to various extreme
sites?

How much genetic information can be dedicated to efficient dispersal
mechanisms?

It has been suggested, for example, that hydrothermal vents would act as infrared
light beacons, and some community members appear to have infrared detectors.
Does this mechanism guide dispersal?

To what extent are stable survival forms of extremophiles different from those that
have been studied?
5. Indicators of biological processes
Microorganisms interact with and also process substantial quantities of materials that,
as a result, acquire characteristics that become signatures of the biological processes that
altered these materials. For example, a sandy sediment surface that was stabilized by a
biofilm might preserve the distinctive surface of that biofilm long after its demise.
Organic molecules can be biosynthesized for light-harvesting, and their distinctive
molecular structures can persist in the sedimentary rock record as legacies of specific
photosynthetic bacteria. Thus careful assays of remnant biological indicators can not
only reveal the former existence of an ecosystem, they can paint a rather detailed picture
of its membership and principal biogeochemical processes.
The following categories of preservable process indicators collectively can offer this
picture:

Cellular morphologies - the traditional evidence of life, recognizable cells and
fragments can offer definitive evidence of former life, yet are often nonspecific
regarding cellular function. A need for a better understanding of morphological
properties (e.g., pleiomorphism) in extreme environments.

Microscale rock textures - the structure and behavior of cells and cell aggregates
can belie their function (e.g., photosynthesis, taxis) and also serve as templates for
mineralogical and sedimentary textures that survive into the sedimentary rock
record. A more systematic understanding of microbial textures in extreme
environments that are amenable to geologic preservation (e.g., hydrothermal
systems).

Community level structures - These represent an aggregation of microscale
textures and their integration into larger-scale structures (e.g., stromatolites, reefs)
that can become even more enduring geological legacies of community structure and
function.

Organic compounds - beyond merely indicating the former existence of life,
structurally distinctive molecules can identify particular ecosystem processes (e.g.,
15


photosynthesis). There is a Need for further development of extremophile
biomarker systematics.
Stable isotopes - Stable isotopic patterns arise by isotopically-selective enzymes
acting upon, for example, networks of carbon pathways in organisms and their
ecosystems. Isotopes thus necessarily reflect the dominant processes and pathways
in a microbial community. Furthermore, isotopic patterns of biological activity
typically survive well in the geologic rock record. There Need for a broader
inventory of measurements of maximum possible isotopic discrimination of carbon,
nitrogen and sulfur by assimilatory and dissimilatory enzymes in extremophiles.
Light from extrasolar systems - To the extent that a distant planet’s atmospheric
composition has been altered by its biosphere, the light reflected from that planet
can telegraph that biosphere’s existence to astronomers. To the extent that distant
biospheres occupy “extreme environments,” reflected starlight might someday
reveal the abundance and diversity of extraterrestrial extremophiles. It might also
enable production of better models of the range of atmospheric compositions in
earth’s pre-oxygenated world.
6. Microbial diversity data
Phylogenetic trees by themselves do not provide information on abundance activity or
function. Efforts need to be directed, in the context of phylogenetic data towards
quantitative assessments, spatial and temporal variability, and functional assessment.
These include:

What are the commonalities and differences within and between specific extreme
habitats?

What is the biological diversity (intraspecific, interspecific, functional) in
particular extreme habitats past and present?

What is the environmental diversity/variability in different extreme habitats?

How does diversity change along environmental gradients that approach biological
limits?

What are indicators of biological activity in terrestrial extreme environments that
could be diagnostic for extraterrestrial life?
7. Evolutionary adaptation to extreme environments
Adaptations that allow organisms to exist in the context of extreme environments are
attained through the acquisition of novel genes. Evolution in extreme environments may
utilize novel or typically rare evolutionary mechanisms. Genetic characterization of
organisms from extreme environments, phylogenetic comparisons across the tree of life
and the analysis of genetic variation through time and space will allow the elucidation of
the mode of evolution in extreme environments. Since the phylogenetic patterns of
single genes are commonly incongruent, genome level analyses are fundamental to these
questions. A balance of coverage over the entire tree of life and the detailed analysis of
several model systems are needed. Informational macromolecules of extremophiles are
16
frequently difficult to extract and isolate from extreme environments so novel approaches
will undoubtedly be required. Input from the engineering community will be especially
valuable in all aspects of the isolation (without contamination) of organisms from
extreme environments. In addition, the adaptations of organisms through time to
extreme environments have not been static. The geological record needs to be used as a
way of determining how, when, and why (the physical environmental context) these
changes have occurred.
Microbes appear to be able to evolve very rapidly, since they have short generation
times, can tolerate high mutation rates, and engage in promiscuous genetic exchange.
Their ability to populate extreme environments affords opportunities to measure these
rates. It raises the question of what maintains identifiable microbial species. One might
expect microbes to show only very limited central tendencies, whereas the few studies on
this topic that have been completed show strong clustering.
Experimental measurements of genomic changes during the relatively short term
‘evolution’ of hydrothermal vents, for example, might illuminate these questions. Such
measurements would require either field or laboratory sampling systems that could be
used over time in either closed or open systems. Complete population profiles of the
communities would need to be developed and complete genomic sequencing information
on selected members obtained over time. Detailed physiological characterization of the
same targeted organisms should accompany the genomic analyses. The null hypothesis
might be that no new traits would evolve, but rather new species would move into the
niche.







What is the genetic and molecular basis of adaptation to extreme environments?
How do molecules/organisms/communities adapt functionally to the harsh
(detrimental) aspects of their environment?
How do molecules/organisms/communities take advantage of opportunities unique
to or created by their environments? (e.g. infrared photosynthesis near smokers,
unusual chemotrophies)
What are the mechanisms that generate these adaptations? (vertical and horizontal
transfer of information, symbioses/syntropy, etc) How do these mechanisms
constrain the limits of life processes?
What is the geologic record of evolution of life processes in extreme
environments?
What geologic traces remain of life processes in extreme environments? How can
the details and generalities of life processes be inferred from the geologic record?
What is the distribution of extreme environments over time? Has this driven
evolutionary transitions between extremophily and mediocrephily? Has the
definition of ‘extreme environments’ changed at the scale of geologic time, and if so
how could this impact evolutionary processes?
8. Possible Foci for Future Research
17
 How does diversity change along environmental gradients that approach biological
limits?
The diversity and extent of life along a stable high temperature thermal gradient
would be examined. The current limit of life at high T in a pure culture is 113C.
However, little is known about the diversity of life at high T, and how it changes in along
thermal gradients above 100C. This proposal seeks to address the question "How does
diversity change along environmental gradients that approach biological limits?"
The following are physical and chemical measurements which will be used in this
research. One of the requirements of our study is that the predominant gradient sampled
is thermal, and not significantly different in terms of chemical composition or other
physical and chemical parameters. It will be necessary to establish, in situ, a suite of
instrumentation to measure and continuously monitor pH, temperature, inorganic ions,
eH, etc. along the gradient. This will engineering and ground truthing chemical and
physical probe development and data logging, and deployment strategies. The biological
process measurement (this requires high temperature methodological development
instrumentation) includes: CO2 fixation, heterotrophic potential, N2 fixation, and Ch4
production. Sampling will include harvest of in situ biomass from solid biofilm
scrapings, as well as colonization slides. Requires instrumentation development and
deployment strategies.
Diversity will be assessed from a variety different levels from intraspecific to functional
levels. The first pass will include characterization of rRNA gene diversity at specific
points along the gradient. Major rRNA groups will be quantified by quantitative pcr
methodologies. Dominant "species clusters" will be identified, and the intraspecific
diversity (microheterogeneity) will be assessed by comparison of rRNA spacer regions.
The research team requires a range of expertise including engineering,
biogeochemistry, microbiology and molecular biology and population biology.
 Microbial communities in permanently ice covered lakes in Antarctica
Several different analogues of extraterrestrial life exist on the continent of Antarctica.
One type of environment, lakes, that are permanently isolated under the Antarctic ice
sheets for millions of years, may be an analogue for potential subsurface aqueous
reservoirs similar to those thought to exist on Europa. Besides first order information
gained on diversity of life on Earth at low temperatures, this project will involve the
development of general methods, procedures, and instrumentation for sampling deep
aqueous reservoirs buried under thick layers of ice -- technology that may be useful in
guiding potential future explorations of Europa. We propose to develop and apply
methods to aseptically recover microbiological samples in waters deep beneath the
Antarctic ice sheets.
 Analysis of microbial communities 1000m below the sea bed
18
Microbial cells have been reported at densities of 1e4/gram of sediment 500 m
below the seabed in a variety of geographic locales. However, most data are either
isolates recovered from these sediments or total cell numbers. A major question is "are
these microorganisms alive and active, or simply preserved after burial?" We propose
here to assess the activity of microbial cells, in situ, 1000m below the sea surface.
Radioisotopic tracer methods, in tandem with an in situ drilling/sampling/injection
system, will be developed and applied to assess the activity of microbial cells in situ.
Methods, Technology, and Infrastructure
1. Methodology
Challenges posed by extreme environments include their remote nature, harsh
conditions, and inaccessibility. Therefore one major issue is identifying methodologies
and techniques that can improve abilities to detect, identify and characterize life in
extreme habitats. Methodologies that need to be developed, improved, or adapted for
use in extreme habitats include sampling, detection, identification, cultivation, in situ
monitoring, and remote sensing. In tandem with increased data collection capabilities,
improvements in data processing and bioinformatics will also be required. There are
specific technological and methodological limitations that would be useful to address in
the study of extreme habitats and organisms. These include:

In situ monitoring (cellular/ecological): Methods for observing microbial
communities on the scale of microns; determining the composition of organisms in
order to interpret and integrate biogeochemical measurements; quantitative methods
for assessing microbial species variability and activities; non-destructive
physicochemical techniques for assessing the energetics of cells and discrete
biochemical processes; metabolic pathway monitoring in genetically engineered
extremophiles; molecular-level probes that indicate biological and chemical
processes during real time measurement

Isolation and cultivation (sustained growth): Generic techniques for the isolation of
microbes from extreme conditions and difficult-to-access environments; robotic
systems; generic techniques for cultivation of key microbes; sustained growth
bioreactors

Biomarker analysis and systematics: an identification/development of suitable
biomarkers indicative of life; an evaluation of these biomarkers for extraterrestrial
utility; and remote-sensing biomarker technology

Modeling: modeling of molecular, biochemical, cellular, multi-cellular and
ecosystem processes
19

Informatics: Information resources (databases) of relevance to major issues and
research thrusts such as extremophile genomes of interest, cellular processes,
extremophile inventory, and metabolic pathways; phylogenetic surveys of natural
microbial diversity having significant numbers of samples (a useful resource and
guide for further studies).
A first order question is simply, what organisms exist (qualitatively and
quantitatively) in any specific habitat. In many habitats only a very small percentage of
microorganisms can be cultivated by traditional means. A recent solution has been to
retrieve and identify phylogenetically informative gene sequences (e.g., rRNA) from total
population DNA, to assess microbial diversity. This is an important approach to
characterizing microbial communities, and necessary in many instances to gain insight
into the composition of naturally occurring microbial communities. The phylogenetic
diversity of a community based on a single gene sequence, however, is of limited use
unless viewed in the context of other studies. Single pass phylogenetic surveys will not
shed light on the nature of extreme habitats or organisms as a stand-alone approach.
However, phylogenetic surveys are a cornerstone for further quantitative, functional, and
temporal assessments of microbial communities associated with extreme habitats.
2. Community building
Because of the inherently interdisciplinary nature of LExEn studies, and especially
investigations of the functional diversity of life and extreme environments, it is
particularly important to bring scientists from the associated fields together for
cross-fertilization (and simply to teach each other the basics of the other fields and their
languages), particularly people in the early stages of their careers.




Team building - The level of effort necessary for thorough survey by molecular
phylogenetic approaches is substantial, and requires focused effort. To place these
efforts in the context of the organisms and environment, team efforts linking
phylogenetic survey approaches with the efforts of geochemists, physicists,
microbial ecologists, physiologists, etc. should be encouraged.
Meetings - short, inexpensive meetings designed to encourage student
participation.
Student training grants or fellowship programs - focused on interdisciplinary
projects or groups (e.g. microbiology with environmental sciences)
LExEn observatories or research stations - to focus a broad range of studies on a
small number of sites so that data can be cross-correlated. Yellowstone National
Park would be an example, perhaps with a few vents specifically targeted. This
could include continuous monitoring and a sample collection service, and a field lab
for LExEn studies.
20
IV. How Does Life Evolve From Pre-Biotic Conditions?
Premises
- Life involves metabolism, reproduction, and biological evolution.
- We are dealing with life that is carbon and water based.
- An extreme environment is understood to be relative to anthropocentric terms.
Specific Research Topics
1. Did life on earth originate in an ‘extreme’ environment?

Did origin of life take place in systems analogous to modern extreme
environments?
 What was the early environment (and range of environments) on early earth?
- Extreme environments such as high temperature hydrothermal systems, deep
subsurface and cold environments and other planetary bodies have all been
proposed as possible analogs for environments that could support an origin of life.
Understanding the early environment that existed prior to and at the time at which
life may have originated on Earth, and the atmospheric and geological processes
that prevailed, is fundamental to testing the theories of how life originated in our
solar system. For example, could the first biological molecules have been
formed, assembled and remained stable under proposed early environmental
conditions?
 What role did impacts play in the origin of life?
- Giant asteroid or planetesimal impacts were a major force on the earliest
history of the Earth, and must have influenced when life originated and the
sustained existence of early life. Potential roles may have involved the supply of
extraterrestrial materials to Earth that could have contributed to the first molecules
of life, the creation of localized extreme high temperature environments, and the
ability of the largest impacts to extinguish life. Additionally, the extent to which
extraterrestrial organic molecules might have contributed to the origin of life
raises the question whether or not some of the first organisms to arise on Earth
were heterotrophs or chemolithoautotrophs.
2. What is the range of extreme environments in which life can originate?

What are the possible mechanisms by which life can originate?
- Includes theoretical and experimental investigations of mechanisms necessary for
the origin of metabolic, reproductive, evolutionary capacities of life--processes that
21
permit concentration of biologically relevant molecules, templates for assembly, and
origin of self-replicating molecules with metabolic capabilities.

What are the physical and chemical prerequisites for the origin of life in extreme
environments and how do these contribute to the formation and persistence of
chemical precursors of life in these environments?
- Early earth differentiation almost certainly resulted in formation of atmosphere
and aqueous components characterized by relatively large concentrations of CO2
and H2, together with reduced N and S compounds. Moreover, the high thermal
flux, active volcanism and connective circulation of “seawater” derived fluids could
bring these components together under the right conditions and for sufficient times
to allow the synthesis of monomeric and polymeric organic compounds, which
could have served as precursors of life on earth. These very same components and
conditions also characterize portions of recent volcanogenic seafloor hydrothermal
systems (on earth and others planets, as well), and might provide modern analogues
and present day clues to the origin and evolution of life on earth. Precursors of life
might include species such as acetate, which could be derived from appropriate
concentrations of CO2(aq) and H2(aq), as follows: 2CO2(aq) + 4H2(aq) =
CH3COOH(aq) + 2H2O. Future research efforts will be needed to assess
unambiguously the role of temperature, pressure, pH and kinetic processes,
involving mineral composition surface areas, to constrain better the formation and
persistence of abiotic compounds leading to the formation of life in extreme
environments.

What is the relationship between interstellar molecules (and cometary molecules)
and the origin of life?
- In living environments, metabolic pathways have been developed to make
available for chemical combination key ingredients that require different
environments to create them, e.g. acids and bases. In the origin of life there is
likely to be a need for such material that a single chemical environment cannot
supply. While it is possible that materials from different planetary environments
can mix, it is also plausible that the missing ingredients come from infall. Such
material seems to be created in dense clouds in interstellar space, frozen together as
ices, and arrives on earth as meteoroid or cometary impacts.

Can an origin of life occur and result in sustained existence in modern extreme
environmental conditions?
- Questions include the identification of environments that have the physical and
chemical conditions conductive to the formation of nucleotides and other molecules
necessary to for the metabolic, reproductive, and evolutionary processes characteristic
of life. What is the fate of these molecules in the modern environment?--are they
absorbed to clays?, are they used for nutrition by existing living organisms before they
can accumulate to the necessary concentration?
22
3.
Where did environments conducive to the origin of life exist?
 What are the geological processes that give rise to such environments on the
planets?
- The major aspects of environments suited for an origin or the continued
existence of life are thought to be liquid water, access to the biogenic elements,
and a source of energy to drive chemical disequilibrium. Any planet that is
geologically active should be capable of providing these. Differentiation,
planetary volcanism, and asteroid or planetesimal impacts, in particular, must
have played a major role in supplying these. And, as geological activity is
strongly tied to planet size, the larger Earth-like planets (Earth, Venus, and Mars
in our solar system) are the most likely places for sustained geological and
biological activity. Understanding the nature of these geological processes, the
underlying processes that control them, and their occurrence on the Earth and
planets is important to understanding the potential mechanisms for an origin of
life.
 What was the actual distribution of such environments on the Earth?
- The history of the Earth's geological processes and of its climate is key to
understanding the ability of life to originate or to exist. In the earliest epochs,
especially, the geologic record contains scant information on the environment, and
there is no record from the time of the origin of life itself. It is important to
determine the abundance of liquid water at the surface, of CO2 and other
greenhouse gases that control temperature in the atmosphere, and of impact- and
volcano-driven hdyrothermal systems, as well as of the influx of materials from
space and of the nature of the chemical environment.
 What is the distribution of planets around stars?
- The frequency of planets is currently best estimated from the frequency of
pre-planetary disks during star formation. The direct or indirect observation of
planets is much harder, and some of the search techniques have tended to fall into
the cracks between NSF and NASA. Particular techniques needing attention are:
- adaptive optics and coronography for the direct imaging of planets
occultation of the star in eclipsing binary systems
- gravitational lensing by planets, which requires more intensive
observations than are currently made
4.
What were the conditions under which life evolved?

Are there extreme environments in existence today that emulate these conditions?
- Central to understanding the early evolution of life is the question of whether
extreme environments now found on Earth could have supported the development
23
of nascent life. Studying the extreme environments of present-day Earth from the
viewpoint of whether they could have supported evolutionary development will
help elucidate possible pathways and mechanisms of evolution. Environmental
parameters of interest might include temperature, pressure, water availability,
energy and carbon sources, and atmospheric conditions.
5. How do fluctuations in physical and chemical environments affect the evolution of
organisms?

Do environmental changes that create extreme environments drive evolution?
- Current evidence suggests that the early Earth was geologically dynamic,
characterized by extreme environmental events. The energy and environmental
conditions resulting from these events may have played a central role in driving
and/or redirecting evolution.

Is short-term environmental change necessary to the origin or evolution of life?
- Severe disruptions to the environment over short time periods may have played
a critical role in dictating the path of evolution. For example, early asteroidal
bombardment may have restricted the evolution and maintenance of life to
deep-sea hydrothermal vents or subsurface environments.

What are the feedback mechanisms between evolution of life and environment?
- In extreme environments, the microbial activity and geochemical environment
are closely associated. For example, biogeochemical processes in extreme
environments may result in significant mineral precipitation, which in turn
influences the geochemical and geological record. Understanding such
interrelationships is important for understanding the evolution of life on Earth.
6. Are there unique genetic features of life in extreme environments?
On the level of populations, we may find that genetic exchange among fairly distantly
related organisms is more (or less, we don't know) common than for microbial
populations in more benign environments. If genetic variation is produced exclusively
by mutation, one would see closely related "clusters" of clones. The genome of
extremophiles may also have characteristic features such as G+C content, etc.

How does life evolve into or out of extremophily?
- It may involve a fairly small number of genetic changes to adapt a microbe to
various extreme environments, or the changes involved may be so widespread that
the descendents of this branch are “locked into” a particular environment.

What features are attributable to common ancestry vs. unique adaptation?
- In this, one is trying to distinguish between convergent evolution (unrelated
organisms with similar phenotypes, due to adaptation to the same environment) vs.
24
organisms having similar phenotypes because they are derived from a common
ancestor and retain those ancestral features.
7. Have extreme environments been central to the evolution of life?
One hypothesis resulting from studies of the evolutionary relatedness of
microorganisms is that early life was thermophilic. Models of a high temperature Earth,
at times when fossilized microbial life is first detected, are consistent with the proposal
that early life evolved in extreme environments. In addition, interplanetary transport of
life would also demand resilience to extreme conditions. It is conceivable then, that
modern day extreme environments harbor forms of life that are similar to ancestral life.
The demands on life in extreme environments might also spawn novel metabolisms that
shed light on the pathways down which evolution has proceeded. The following
questions represent the sort of guiding questions under which specific research projects
might be developed.

What do gene phylogenies tell us about the early history of life?

Are extreme environments reservoirs of ancestral-like life forms?
 What role have extreme environments played in the origin of new metabolic
pathways?
Methods, Technology, and Infrastructure
1. Encourage theoretical development
It is recommended that efforts be made to obtain the wide range of thermodynamic
and kinetic data needed to assess unambiguously mechanisms and processes involving the
formation of monomeric and polymeric organic compounds, which are needed to
understand better how these species contribute to the formation of the more complex
organic molecules and the origin of life in extreme environments. These investigations
should attempt to take explicit account of the potential interactions between microbes and
their environment.
2. Technology development

Remote sensing
- Sensing life resources: Remote communication of evidence will be by
absorption or emission of electromagnetic radiation, and the sensing will be by
spectroscopy. In sensing life resources, e.g. interstellar or cometary molecules,
the significant chemicals are usually well-known, e.g. amino acids, but their
spectral signatures in the radio region are generally unknown so lab studies are
needed.
- Sensing life and death products: Life depends on the conversion of a high
degree of order in a heat or light flow into a chemical separation of living matter
25
and waste products. Competition drives organisms to highly efficient and near total
use of environment chemical resources.
Similarly death results in the
decomposition of matter far out of balance with its environment. When these
products are released to the atmosphere, they introduce detectable compounds far
from chemical equilibrium, some of which destroy each other on a short time
scale. This will be a detectable signature of a planet with life. Here the IR spectra
of key simple molecules are well known. The need instead is to identify key
metabolic pathways of life and decay, particularly of archaea under anaerobic
conditions.

In-situ sensing
- There is a great need to develop techniques for continuous remote monitoring of
the biological, biochemical, chemical and physical characteristics of extreme
environments. These developments will require nurturing of long-term efforts
between technologists, analytical chemists and end users of the information.

Laboratory reactors that simulate extreme environments
- Laboratory studies of possible abiotic synthesis pathways and the incubation of
extremophiles under controlled conditions will require continued development of
physical-chemical conditions found in extreme environments. These systems
should be developed in close collaboration with the monitoring sensor program.
4. Community building




Establish necessary infrastructure for continuous monitoring and sampling at
observatories and for communication of results from these sites
Support interdisciplinary proposals and workshops
Support graduate students, postdoctoral fellows and sabbatical leaves for
cross-disciplinary research
Identify a few sites for intensive investigation (Extreme Environment
Observatories)
- offers potential for interpreting biological, chemical and physical data in light
of other information - collected from same site
- continuous monitoring of environments
- development of principles that could be tested elsewhere
26
Appendix A
LIFE IN EXTREME
ENVIRONMENTS
(LExEn)
Announcement of Opportunity and
Special Competition for FY 1998
DIRECTORATE FOR BIOLOGICAL SCIENCES
DIRECTORATE FOR ENGINEERING
DIRECTORATE FOR GEOSCIENCES
DIRECTORATE FOR MATHEMATICAL AND PHYSICAL SCIENCES
OFFICE OF POLAR PROGRAMS
DEADLINE DATE: January 15, 1998
NATIONAL SCIENCE FOUNDATION
27
Introduction
Life flourishes on Earth in an incredibly wide range of
environments, from high-salt deserts to volcanoes to polar ice.
These environments may be analogous to the harsh conditions that
exist now, or have existed, on other planets. The study of
microbial life-forms and the extreme environments in which they
exist here on Earth can provide important new insights into how
organisms form and adapt to diverse environments. This knowledge
will provide the basis for detecting and understanding the life
forms that may exist beyond our own planet, and for developing
useful new products and processes.
The Directorates for Biological Sciences (BIO), Engineering
(ENG), Geosciences (GEO), Mathematical and Physical Sciences
(MPS), and the Office of Polar Programs (OPP) of the National
Science Foundation (NSF) announce an opportunity to enhance
knowledge about “Life in Extreme Environments” (LExEn) through
highly interdisciplinary, integrated research activities.
LExEn Interdisciplinary Research Program
The LExEn interdisciplinary research program will explore the
relationships between microorganisms and the environments
within which they exist, with a strong emphasis upon those
life-supporting environments that exist near the extremes of
planetary conditions. In addition, the LExEn program will
explore planetary environments in our own solar system and
beyond to help identify possible sites for life elsewhere.
Research is required to enhance understanding of the
microbial systems on Earth, particularly with respect to their
diversity and the mechanisms that allow microbes to survive and
alter extreme environments.
Examples of relevant topics
include:




studies to discover, quantify, culture, preserve, and
analyze microorganisms from extreme environments;
research
on
the
diversity,
ecology,
physiology,
biochemistry, genetics and evolutionary history of
microbes from extreme environments within the context of
the diversity of conditions found in those environments
and/or their possible utility in biotechnology;
the identification of unusual or even unique chemical
compounds and their specific roles in survival strategies;
and
paleobiological studies of microbial life on Earth,
including efforts which improve understanding of the
products of life that are preserved in the geological
record.
Because of the importance of the interactions between living
organisms and their habitats, it is necessary to achieve
improved understanding of present-day or past extreme
environments that support or have supported life.
28
Examples of relevant topics include studies designed to
explore and fully characterize significant examples of extreme
environments in order to understand the active physical,
chemical, biological and geological processes that determine
their characteristics and result in their ability to support
unusual microbial life. In order to provide insights into the
possibility of life beyond our own planet, research is also
needed to characterize the environments of planets in the solar
system and beyond and to understand the commonalities of their
formation and evolution. Examples of relevant topics include
studies of the formation of Earth, other planets and their
satellites; remote sensing of planets and their atmospheres;
studies of interstellar grains and meteorites to establish
criteria for the presence of biogenic substances; and research
on the biogeochemical effects of microbes on their environments
on Earth to better design tests for life on other planets.
FY 1998 Special Competition
NSF is highlighting its interest in LExEn through a special
competition as described below. Proposals must be received at
NSF no later than close of business on January 15, 1998. Review
and processing of proposals require approximately six months.
For the purposes of this announcement "extreme" refers to
environments found today on Earth which have attributes that are
similar to those that exist on other planetary bodies (at present
or in the past), or to those that are postulated to have existed
on Earth at the dawn of life. Such environments might include
those associated with hydrothermal systems, sea ice and ice
sheets, anoxic habitats, hypersaline lakes, high altitude or
polar deserts, or man-made environments such as those created
for industrial processes.
It is intended that this Special Competition will focus on
hypothesis-driven projects that show potential for establishing
a foundation for future innovative research related to LExEn.
Because of the importance of the interactions between living
organisms and the extreme environments within which they exist,
studies are particularly encouraged that cross disciplinary
boundaries and foster collaborative investigations.
For this Special Competition, projects should couple one or
more of the research areas described in the previous section with
the development and application of one or more of the following:

methods to isolate and culture microbes found in extreme environments;

methods to study these microbes in their natural habitats and to describe their adaptive strategies
from the molecular to the ecological level;

technologies for non-contaminating sample recovery;

sensors and sensing techniques to probe extreme environments on Earth or other planets;

methods to study ancient microbial life and paleo-environmental conditions on Earth; and
29

methods to investigate the potential for habitable environments on other planets (including theory
and modeling).
Research projects of 2-5 years duration are appropriate.
Proposals that are predominantly for the purchase of available
equipment or instrumentation are not appropriate for this
competition. Funds available to support projects under this
Special Competition are expected to total approximately $6
million. NSF anticipates making approximately 20 awards in
fiscal year 1998 depending on the quality of the proposals
received and the availability of funds.
Preparation and Submission of Proposals for the FY 1998 Competition
Proposals submitted in response to this Announcement of
Opportunity will be accepted from colleges, universities, and
other non-profit institutions in the United States. Proposals
involving collaboration with researchers and facilities of
other countries or international groups are welcome, provided
support is requested only for the U.S. portion of the
collaborative effort.
Proposals should be prepared and submitted in accordance with
the NSF Grant Proposal Guide (GPG) NSF 98-2 and the Proposal
Forms Kit NSF 98-3 (replacing NSF 95-27 and NSF 95-28). For
proposals involving field work in Antarctica, guidelines
described in the “Antarctic Research Opportunities and Proposal
Guide” NSF 96-93 should be followed. These documents can be
accessed through the NSF Home Page (http://www.nsf.gov/) or
you may request hard copies at no cost from:
NSF Clearinghouse
P.O. Box 218
Jessup, MD 20794-0218
TEL: 301-947-2722
e-mail: pubs@nsf.gov
Proposers requiring the use of a UNOLS ship or submersible must
submit a ship request form to NSF’s Division of Ocean Sciences
as well as the UNOLS office and the operator of any requested
ship or ships.1
Proposals will be subjected to initial screening for the
requirements in the GPG and will be returned without review or
advance notification if deficiencies are found. Proposals will
NOT be forwarded to other programs if found to be inappropriate
for this competition. Proposals submitted in response to this
announcement must be received at NSF no later than close of
business on January 15, 1998.
Group and collaborative proposals involving more than one
institution MUST be submitted as a single administrative package
from one of the institutions involved. Multiple submissions
1
Electronic ship request forms are available on the UNOLS Home Page (http://www.gso.uri.edu/unols/experiments/
experiments.html). Printed copies of the form may be obtained from NSF Division of Ocean Sciences or the UNOLS office (tel:
401-874-6825; email: unols@gsosun1.gso.uri.edu).
30
will not be accepted. (The proposal may be split into separate
awards if the project is recommended for support.) The package
should include one project summary, one table of contents, one
project description, one section for references, and one copy
of special information and appendices as specified in GPG
section II.D.10-11. Additionally, the package should include,
for each university and its PIs/co-PIs, a signed cover sheet,
budget pages and explanation, results from prior NSF support (up
to 2 pages per person), biographic sketches (up to 2 pages per
person), current and pending support for each PI/co-PI, and
facilities and other resources unique to each institution.
LExEn should be referenced in the upper left corner of the
proposal cover sheet as the NSF organizational unit. The
solicitation number is NSF 97-157. An original and 20 copies of
the proposals should be sent to:
Announcement No. NSF 97-157
Proposal Processing Unit
National Science Foundation
4201 Wilson Blvd., Room P-60
Arlington, VA 22230
Proposals may also be submitted electronically via Fastlane.
For information, contact FastLane user support services (tel:
703-306-1142; email: fastlane@nsf.gov).
Proposal Review
Proposals will be evaluated by ad hoc mail and panel review
in accordance with established Foundation procedures and the
criteria described below. It is anticipated that each review
panel will have expertise in the fields of astronomy, planetary
sciences, chemistry, geochemistry, biology, atmospheric
sciences, earth sciences, ocean sciences, polar sciences, and
engineering.
Proposals submitted in response to this program announcement
will be subject to the NEW merit review criteria approved by the
National Science Board on March 28, 1997 (NSB97-72).2 The new
merit review criteria are:
*
What is the intellectual merit and quality of the proposed
activity?
For example, how important is the proposed activity to
advancing knowledge and understanding within its own field and
across different fields? How well qualified is the proposer
(individual or team) to conduct the project? (If appropriate,
reviewers may also comment on the quality of prior work.) To what
extent does the proposed activity suggest and explore creative
2
For additional information on NSF’s new merit review criteria, see the Merit Review Task Force Final Report on the NSF Home
Page at http://www.nsf.gov/cgi-bin/getpub?nsfmr975.
31
and original concepts? How well conceived and organized is the
proposed activity? Is there sufficient access to resources?
*
What are the broader impacts of the proposed activity?
For example, how well does the activity advance discovery and
understanding while promoting teaching, training, and learning?
How well does the proposed activity broaden the participation
of
underrepresented
groups
(e.g.,
gender,
ethnicity,
disability, geographic, etc.)? To what extent will it enhance
the infrastructure for research and education, such as
facilities, instrumentation, networks, and partnerships? Will
the results be disseminated broadly to enhance scientific and
technological understanding? What may be the benefits of the
proposed activity to society?
In addition to these generic review criteria, an additional
criterion in the evaluation process for this competition will
be the potential interdisciplinary synergism among the various
research components. For this reason, the ways in which the
various projects proposed will be divided and coordinated among
the different researchers needs to be carefully thought out and
described.
Grant Administration and Conditions
Grants awarded as a result of this announcement will be
administered in accordance with the terms and conditions of NSF
GC-1 (10/95) or FDP-III (7/1/96), Grant General Conditions.
Copies of these documents are available from the NSF online
document
system:
http://www.nsf.gov/cgi-bin/
pubsys/browser/odbrowse.pl. More comprehensive informa-tion
is contained in NSF 95-26, Grant Policy Manual (7/95), for sale
through the Superintendent of Documents, Government Printing
Office, Washington, D.C. 20402.
FY 1999 and Beyond
The nature of future LExEn special competitions is still under
consideration.
Future announcements will be posted
electronically with hard copies available upon request. If you
would like to be alerted to the posting of these announcements,
please send a message to “listmanager@nsf.gov” with the command
“subscribe nsflexen <your full name>” in the text of the message
(the subject line is ignored). Your e-mail address will be
extracted from the “From:” field of your request.
For More Information
This announcement, as well as other LExEn-related
information, is available on-line at http://www.nsf.gov/
home/crssprgm/lexen/start.htm.
Should you wish to receive a
hard copy of this announcement, please contact the NSF
publications office (tel: 301-947-2722; email: pubs@nsf.gov).
32
If you have questions or require further information,
contact:
Division of Environmental Biology
Charles O’Kelly, (703)306-1481, cokelly@nsf.gov
Division of Molecular and Cellular Biosciences
Philip Harriman, (703) 306-1439, pharrima@nsf.gov
Division of Astronomical Sciences
Vernon Pankonin, (703) 306-1826, vpankoni@nsf.gov
Division of Chemistry
George Rubottom, (703)306-1851, grubotto@nsf.gov
Division of Bioengineering and Environmental
Systems
George Vermont, (703) 306-1318, gvermont@nsf.gov
Division of Atmospheric Sciences
Jarvis Moyers, (703)306-1523, jmoyers@nsf.gov
Division of Earth Sciences
Chris Maples, (703)306-1551, cmaples@nsf.gov
Division of Ocean Sciences
Phillip Taylor, (703)306-1587, prtaylor@nsf.gov
David Epp, (703)306-1586, depp@nsf.gov
Office of Polar Programs - Antarctic Research Section
Linda Duguay, (703) 306-1033, lduguay@nsf.gov
Office of Polar Programs - Arctic Research Section
Douglas Siegel-Causey, (703) 306-1030,
dsiegel@nsf.gov
The National Science Foundation provides awards for
research and education in the sciences and engineering.
The awardee is wholly responsible for the conduct of such
research and preparation of the results for publication.
The Foundation, therefore, does not assume responsibility
for the research findings or their interpretation.
The Foundation welcomes proposals from all qualified
scientists and engineers and strongly encourages women,
minorities, and persons with disabilities to compete fully
in any of the research and education related programs
described here. In accordance with federal statutes,
regulations, and NSF policies, no person on grounds of race,
color, age, sex, national origin, or disability shall be
excluded from participation in, be denied the benefits of,
or be subject to discrimination under any program or
33
activity receiving financial assistance from the National
Science Foundation.
Facilitation Awards for Scientists and Engineers with
Disabilities (FASED) provide funding for special assistance
or equipment to enable persons with disabilities
(investigators and other staff, including student research
assistants) to work on NSF projects.
See the program
announcement or contact the program coordinator at (703)
306-1636.
The National Science Foundation has TDD (Telephonic
Device for the Deaf) capability, which enables individuals
with hearing impairment to communicate with the Foundation
about NSF programs, employment, or general information. To
access NSF TDD, dial (703) 306-0090; for FIRS,
1-800-877-8339.
Privacy Act and Public Burden
The information requested on proposal forms is solicited
under the authority of the National Science Foundation Act
of 1950, as amended. It will be used in connection with the
selection of qualified proposals and may be disclosed to
qualified reviewers and staff assistants as part of the
review process; to applicant institutions/grantees; to
provide or obtain data regarding the application review
process, award decisions, or the administration of awards;
to government contractors, experts, volunteers, and
researchers as necessary to complete assigned work; and to
other government agencies in order to coordinate programs.
See
Systems
of
Records,
NSF
50,
Principal
Investigators/Proposal File and Associated Records, and
NSF-51, 60 Federal Register 4449 (January 23, 1995),
Reviewer/Proposal File and Associated Records, 59 Federal
Register 8031 (February 17, 1994).
Submission of the
information is voluntary.
Failure to provide full and
complete information, however, may reduce the possibility
of your receiving an award.
The public reporting burden for this collection of
information is estimated to average 120 hours per response,
including the time for reviewing instructions.
Send
comments regarding this burden estimate or any other aspect
of this collection of information, including suggestions
for reducing this burden, to Gail A. McHenry, Reports
Clearance Officer, Information Dissemination Branch,
National Science Foundation, 4201 Wilson Boulevard, Suite
245, Arlington, VA 22230.
34
Appendix B
LExEn Workshop Agenda
Wednesday, June 17- Friday, June 19, 1998
National Science Foundation
4201 Wilson Blvd., Arlington VA 22230
Wednesday, June 17, 1998
9:00 a.m.
Steering Committee Meets, Room 730
1:00 p.m.
Workshop Opens- Chaired by Mike Purdy
Welcome and Introductions
LExEn Program overview
Definition and discussion of workshop goals
2:00 p.m.
Short Presentations begin - 18 at 5 min each Chaired by Jean Brenchley
3:30 p.m.
Break
3:45 p.m.
Short Presentations continue - 18 at 5 min each Chaired by Dave Karl
5:15 p.m.
Discussion lead by John Delaney
6:30 p.m.
Reception
Thursday, June 18, 1998
7:00 a.m.
Steering Committee Breakfast
8:00 a.m.
Coffee and Doughnuts
8:30 a.m.
Establish Working Groups and define charges
9:30 p.m.
Break
10:00 a.m.
Working Groups meet
12:30 p.m.
Lunch (Steering Committee meets)
1:30 p.m.
Working Groups meet
5:00 p.m.
Plenary Session: Working Groups report
6:00 p.m.
Adjourn
Friday, June 19, 1998
8:00 a.m.
Coffee and Doughnuts
8:30 a.m.
Writing and Working Group Meetings as required
11:00 a.m.
Plenary Session
35
Appendix C
LExEn Workshop Participants
Jean Brenchley, Pennsylvania State University, jeb7@psu.edu
Jim Brown, North Carolina State University, jwbrown@mbio.ncsu.edu
Doug Clark, University of California, Berkeley, clark@cchem.berkeley.edu
Emily CoBabe, University of Massachusetts, ecobabe@geo.umass.edu
Jim Cowen, University of Hawaii, jcowen@soest.hawaii.edu
John Delaney, University of Washington, jdelaney@u.washington.edu
Ed Delong, Monterey Bay Aquarium Research Institute, delong@mbari.org
Dave DesMarais, NASA, ddesmarais@mail.arc.nasa.gov
Kathleen Duncan, University of Tulsa, BIOL_KED@centum.utulsa.edu
Jim Holden, University of Georgia, jfholden@arches.uga.edu
John Holloway, Arizona State University, john.holloway@asu.edu
Julius Jackson, Michigan State University, jhjacksn@pilot.msu.edu
Bruce Jakosky, University of Colorado, jakosky@orion.colorado.edu
Dave Karl, University of Hawaii, dkarl@soest.hawaii.edu
Marv Lilley, University of Washington, lilley@ocean.washington.edu
Douglas N.C. Lin, University of California, Santa Cruz, lin@ucolick.org
Barry Marrs, Fairville Products, Inc., barrym4212@aol.com
Betsy McLaughlin-West, Rutgers University New Brunswick, betsy@koros.rutgers.edu
Robert McMahon, University of Wisconsin, mcmahon@chem.wisc.edu
Anna-Louise Reysenbach, Rutgers University, alr@imcs.rutgers.edu
Thomas Schmidt, Michigan State University, tschmidt@pilot.msu.edu
Jeff Seewald, Woods Hole Oceanographic Institution, jseewald@whoi.edu
Bill Seyfried, University of Minnesota, wes@maroon.tc.umn.edu
Everett Shock, Washington University, shock@zonvark.wustl.edu
Loren Smith, University of Southern California, lhsmith@mizar.usc.edu
Lew Snyder, University of Illinois, snyder@astro.uiuc.edu
Kevin Sowers, University of Maryland, sowers@umbi.umd.edu
36
(LExEn Workshop Participants continued)
Diane Stoecker, University of Maryland, stoecker@hpel.umd.edu
Ron Swanson, Diversa Corporation, rswanson@diversa.com
Patrick Thaddeus, Harvard University, pthaddeus@cfa.harvard.edu
James Tiedje, Michigan State University, tiedjej@pilot.msu.edu
Meg Tivey, Woods Hole Oceanographic Institution, mktivey@whoi.edu
Jack Welch, University of California, Berkeley, wwelch@astro.berkeley.edu
Nick Woolf, University of Arizona, nwoolf@as.arizona.edu
Art Yayanos, University of California, San Diego, ayayanos@ucsd.edu
Oskar Zaborsky, Hawaii Natural Energy Institute, ozabo@hawaii.edu
37
Appendix D
Some LExEn Questions
(prepared for the workshop attendees by Hugh Van Horn)
Can life originate in more than one way?
What is the range of diversity of living organisms?
What is the range of carbon and energy pathways in living organisms?
How can we reconstruct metabolism from DNA sequences?
How large a piece of DNA is necessary to identify clustering?
Can a single gene have more than one function in different organisms?
How can gene fragments be assigned to specific organisms?
How do time changes in physical/chemical environments affect the evolution of
organisms?
How will high-bandwidth data links affect scientific research, especially in
remote/hostile/extreme environments?
Why might Mars or Europa have life or interesting prebiotic chemistry?
What locations might have extant or extinct life?
What would be a good indicator for the existence of life?
What metabolites might provide evidence for the (past) existence of life?
What's out there on Earth?
What is necessary to cultivate “uncultivatable” organisms in the lab?
How does population density affect organisms?
What are the environmental limits to life?
To what extent do extreme terrestrial environments represent possible extraterrestrial
environments?
To what extent can extreme environments be engineered in the lab?
38
What microorganisms can survive cyclic exposure to extreme environments?
How does the fossil record reveal the history of evolution?
What are physical/chemical tracers of past life?
How interconnected are terrestrial environments?
How can evolutionary forces be understood within their ecosystems?
What role does gene transfer play in the evolution of organisms?
What are the "primary producers" in different ecosystems?
How can organisms be studied in their natural environments?
Can microorganisms be usefully modified by microprobes?
How does an organism become an extremophile? (Or vice versa?)
How do extremoophile populations adapt?
How are extremophile communities organized?
Is the rate of evolution faster in extremophiles?
How quickly can microbes adjust to rapidly changing environments?
What is the significance of subseafloor hyperthermophiles?
What are the intracellular energetics of hyperthermophiles?
Are there unique chemical signatures of living organisms?
How are different microbial organisms interdependent?
How does environment affect the organism, and how do organisms affect the
environment?
Can we produce extreme environments in the lab?
Is there liquid water in planets and satellites?
39
Can dynamical evolution of planetary/satellite systems produce environments capable of
supporting life?
Why is cryptobiotic soil clumpy?
How dry can you get and still have life?
What are the important energy sources for life in extreme environments?
What energy sources do we expect to be important on other planets?
"Who's there, and what are they doing?"
Is there a Europan ocean; if so, what is it like?
Are there biologically relevant molecules in interstellar clouds? Comets? Meteorites?
Planetary atmospheres?
What chemical processes produce biologically relevant species?
What were the early atmospheric conditions for life?
Are there isotopic mechanisms capable of distinguishing the metabolic processes of fossil
organisms?
How can we understand the patterns of microbial diversity in extreme environments?
How did life originate?
What processes of abiotic synthesis produced the chemical precursors of life?
When did "novel" metabolic processes arise?
How might astronomical events (e.g., comet/meteorite impacts) have affected the
evolution of life?
What is the relationship between interstellar organic molecules and the origin of life?
What are the abundances of biologically relevant molecules in interstellar space?
What is the origin of complex interstellar molecules?
What strategies can enable us to discover and characterize novel microbes?
How do organisms sustain metabolic activity in extreme environments?
40
How can "consortia" best be studied in the laboratory?
How important is endosymbiosis?
What is the relation between the genomic "tree of life" and evolution?
What organisms live at very cold temperatures?
Are they active in situ at, e.g., -12C?
What adaptive strategies do they have?
How long can viable organisms live at very low temperatures?
How can we sample material from beneath deep ocean vents?
What are the spectroscopic signatures of life in planetary atmospheres?
What are the limits of habitable environments, e.g., in P, T?
Is liquid water necessary for life?
What is the spectrum of environments in which life can originate?
How did protein families and subunits of cells originate?
What prebiotic processes are relevant to the origin of life?
What determines the limits to life?
How does the fossil record shed light on the limits to life?
What processes allow life in extreme environments?
What prebiotic conditions exist in extreme environments?
41