Support for research activity focused on Life in Extreme Environments (LExEn) was formally announced by the National Science Foundation on January 21, 1997. The principal objective of the program is to enhance knowledge about life on earth by exploring the full spectrum of microbial ecology in extreme environments through interdisciplinary research. A key premise is that deep understanding of certain earth-bound microbial systems would provide important insights into life-sustaining processes and the origin of life on our own planet, while illuminating the search for life in other planetary environments.

LExEn activity is intended to catalyze scientific collaboration across a spectrum of disciplines. It encourages development of technologies to enable remote sampling and sensing of life forms and their metabolites. LExEn also focuses on diversity, ecology and physiological capabilities of microbes in order to increase our knowledge about their evolutionary history within seemingly hostile environments on Earth.

To engage the broadest possible community in this activity, NSF convened a Workshop in June 1998, to explore future directions of the overall LExEn Research Effort. The workshop strategy was to engage participants in wide-ranging dialogue focused on three Primary Questions. Each working group focused on a single question with the charge of developing an Investigative Strategy while providing a recommended framework to nurture integrative research activities arising from the strategy. The questions are:

• What conditions determine the limits of life?
• What is the functional diversity of life?
• How does life evolve from pre-biotic conditions?

The working groups were urged to maintain balance between studies of organisms and studies of the key environments, to optimize benefits of interdisciplinary approaches, to promote discovery as well as hypothesis-testing in this new research arena, and to articulate infrastructural elements required to achieve essential objectives.

An extreme environment is one where physical and chemical conditions approach or exceed the tolerances for life. This designation is anthropocentric and although some organisms thrive under extreme conditions, many extremophiles cannot compete successfully, or even survive outside of these extreme habitats. Our basic premise is that understanding life processes in extreme environments will allow constraints to be placed on the limits of life as we know it.

Specific Research Topics explored by this group included lists of questions and comments focused on:

1. The forms of energy required to support life i.e. sources of light, chemical gradients, and other conditions that induce electron transfer;
2. The tolerances that influence limits to the dispersal and distribution of life i.e. temperature, pressure, water activity, redox conditions, pH, salt, nutrients, and trace elements;
3. The roles of such factors as cell size, wall structure and thickness, and enzyme-substrate channeling in determining the success or failure of adaptation strategies to diverse habitats;
4. Assessment of diverse resources essential to life i.e. water, C, N, P, enzymes, and trace metals in extreme environments.

Extreme habitats, by their very nature, are not well characterized. An in depth study of the functional diversity of organisms in extreme environments requires collection and integration of detailed information from different hierarchical levels. These levels 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 both habitats and organisms, "what they're doing" in terms extracting a living from the surroundings, and, "how they’re doing it", in terms of specific adaptations and properties of life within any particular habitat. Community, organismal and molecular processes will be studied using bioenergetics, uptake of substrates, physiology, community interactions, preservable indicators of biological processes, and evolutionary adaptation of metabolic processes to extreme environments.

Specific Research Topics explored by this working group addressed the following issues: physiology and structure, group and species interaction, stress responses, dispersal, indicators of biological processes, microbial diversity data, evolutionary adaptation to extreme environments, and possible foci for future research.

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. These features include physiological and structural adaptations required for basic processes including growth, reproduction, and cell maintenance. Research on these topics requires identification of metabolic pathways and mechanisms of control at both the biochemical and genetic levels.
2. Group and species interactions - The degree to which species interactions and community function in extreme environments differ from those interactions and functions in more benign environments is not known. 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. 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.
3. Stress Responses - Stress responses to be examined: heat or cold shock, pressure stress, osmotic or nutrient deprivation, pH and Eh variation, the availability of water, and dormancy/encystment. Do extremophiles possess novel mechanisms for adapting to environmental stress?
4. Dispersal - Extreme environments are frequently patchy, ephemeral and unpredictable. This structure requires organisms to evolve novel mechanisms to allow persistence during times of environmental change or to promote colonization of new areas. The ecological tolerance of these organisms may be extremely broad, or there may be special adaptations to allow persistence in a resting state.
5. Indicators of biological processes - Microorganisms can produce materials that become signatures of life processes. Assays of remnant biological indicators can reveal the former existence of an ecosystem and can paint a detailed picture of its membership and principal biogeochemical processes. Categories of preservable process indicators include cell morphologies, microscale rock textures, community level structures, organic compounds, and stable isotopes.
6. Microbial diversity data - Efforts must be directed, in the context of phylogenetic data toward quantitative assessments, spatial and temporal variability, and functional assessment.
7. Evolutionary adaptation to extreme environments – Life in extreme environments may utilize novel or rare evolutionary mechanisms. Since the phylogenetic patterns of single genes are commonly incongruent, genome level analyses are fundamental to these questions. The geological record needs to be used as a way of determining how, when, and why changes have occurred. Microbes appear to be able to evolve very rapidly, can tolerate high mutation rates, and engage in promiscuous genetic exchange. Their ability to populate extreme environments affords opportunities to measure these rates. Complete population profiles should be developed and genomic sequencing information on selected members should obtained.

This working group felt the need to establish a set of definitions and assumptions in order to focus and constrain the discussions. They approached the task of exploring pre-biotic to biotic evolution by identifying several premises:

• Life involves metabolism, reproduction, and biological evolution;
• The discussions below address life that is carbon and water-based; and
• An extreme environment is understood relative to an anthropocentric perspective.

Specific discussion points are introduced by a single major question followed by sub-questions that are derived from the major question:

1. Did life on earth originate in an extreme environment? Did origin of life take place in systems analogous to modern extreme environments? What were the environments on early earth? What role did impacts play in the origin of life?

2. What is the range of extreme environments in which life can originate? 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? What is the relationship between interstellar molecules (and cometary molecules) and the origin of life? Can an origin of life occur and result in sustained existence in modern extreme environmental conditions?

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? What was the actual distribution of such environments on the Earth? What is the distribution of planets around stars?

4. How do changes in physics and chemistry affect the evolution of organisms? Do environmental changes that create extreme environments drive evolution? Is short-term environmental change necessary to the origin or evolution of life? What are the feedback mechanisms between evolution of life and environment?

5. Are there unique genetic features of life in extreme environments? How does life evolve into or out of extremophily? What features are attributable to common ancestry vs. unique adaptation?

6. Have extreme environments been central to the evolution of life? 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?

Challenges posed by extreme environments include their remote nature, harsh conditions, and inaccessibility. Methodologies to be developed, improved, or adapted for uses in extreme habitats include sampling, detection, identification, cultivation, in situ monitoring, and remote sensing. The ability to reproduce extreme environments in the laboratory for in depth studies of extremophile physiology is a key component in developing durable models of the entire habitat suite of interest. Increased data collection capabilities, improvements in data processing and bioinformatics will also be required.

Theoretical modeling of entire systems should be encouraged in part by investing in a wide range of thermodyamic and kinetic data. Such information is essential to assess unambiguously the formation of monomeric and polymeric organic compounds, to better illuminate how these species might contribute to the formation of more complex organic molecules leading to the origin of life.

Recommendations were made to develop a sufficiently coherent group of researchers to ensure the highest quality of scientific interplay among disciplines that have not traditionally interacted. These would include, but not be limited to, microbiology, planetary science and astronomy, geology, geochemistry, paleontology, molecular biology and engineering. It is important to bring scientists from the associated fields together for cross-fertilization and mutual instruction on the basics of other fields. This is particularly true of people in the early stages of their careers. Mechanisms posed to accomplish this end include the following: short meetings and cross-disciplinary workshops; LExEn observatories or research stations; infrastructure for continuous monitoring and sampling at observatories and for communication of results from these sites; resource allocation to collaborative projects; support graduate students, postdoctoral fellows and sabbatical leaves for cross-disciplinary research; resources for small grants to exploratory research (SGER) grants; web page development and maintenance.