5

Conclusions and Recommendations

INTRODUCTION

Convened by the Committee on the Origins and Evolution of Life, the Workshop on Life Detection Techniques presented an opportunity for a multidisciplinary group of experts ranging from laboratory biologists to planetary scientists to consider the current state of detection technologies and sampling strategies. Detection of extant and extinct extraterrestrial life, in situ and in the laboratory, was discussed, as were assays of terrestrial microorganisms for the purpose of gauging spacecraft cleanliness. The committee's goal was not to select a particular technique or techniques for use in detection in each case, but rather to identify key issues that must be addressed in the course of developing sample-return and in situ detection missions. Table 5.1 provides a list of biosignatures and their applicability to detecting evidence of extinct or extant life or to detecting terrestrial biological contaminants on spacecraft or in martian samples. These techniques would also have some applicability to the detection of novel life that may have a biochemistry somewhat different from that of terrestrial life. Most of these biosignatures, however, are based on the assumption that putative extraterrestrial life would be carbon based, would have structures that could be recognized as evidence for life, and would utilize available energy sources that are known to support terrestrial life. While Table 5.1 lists biosignatures indicative of extant and potentially viable life, it does not cover all of the methods available that could provide evidence for the growth, metabolic activity, and physiological potential of viable organisms.

Table 5.2 provides an index of the biosignature-measuring techniques presented at the workshop, the properties they measure, their sensitivity, their limitations, and the need for future developments. The table includes techniques that were not covered in the workshop presentations, but whose inclusion here in summary form provides a comprehensive basis for comparing the approaches.

DETERMINING IF LIVING ORGANISMS ARE ON A SPACECRAFT BEFORE LAUNCH

The most strikingly definitive result evident from the workshop is the dramatic improvement in laboratory techniques designed to detect terrestrial organisms, with principal application to spacecraft sterilization and, hence, planetary protection issues relating to forward contamination.

Recommendations regarding specific sterilization techniques and levels of sterilization to avoid contamination of other planetary bodies go beyond the original purview of the workshop and this report. From the point of view



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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques 5 Conclusions and Recommendations INTRODUCTION Convened by the Committee on the Origins and Evolution of Life, the Workshop on Life Detection Techniques presented an opportunity for a multidisciplinary group of experts ranging from laboratory biologists to planetary scientists to consider the current state of detection technologies and sampling strategies. Detection of extant and extinct extraterrestrial life, in situ and in the laboratory, was discussed, as were assays of terrestrial microorganisms for the purpose of gauging spacecraft cleanliness. The committee's goal was not to select a particular technique or techniques for use in detection in each case, but rather to identify key issues that must be addressed in the course of developing sample-return and in situ detection missions. Table 5.1 provides a list of biosignatures and their applicability to detecting evidence of extinct or extant life or to detecting terrestrial biological contaminants on spacecraft or in martian samples. These techniques would also have some applicability to the detection of novel life that may have a biochemistry somewhat different from that of terrestrial life. Most of these biosignatures, however, are based on the assumption that putative extraterrestrial life would be carbon based, would have structures that could be recognized as evidence for life, and would utilize available energy sources that are known to support terrestrial life. While Table 5.1 lists biosignatures indicative of extant and potentially viable life, it does not cover all of the methods available that could provide evidence for the growth, metabolic activity, and physiological potential of viable organisms. Table 5.2 provides an index of the biosignature-measuring techniques presented at the workshop, the properties they measure, their sensitivity, their limitations, and the need for future developments. The table includes techniques that were not covered in the workshop presentations, but whose inclusion here in summary form provides a comprehensive basis for comparing the approaches. DETERMINING IF LIVING ORGANISMS ARE ON A SPACECRAFT BEFORE LAUNCH The most strikingly definitive result evident from the workshop is the dramatic improvement in laboratory techniques designed to detect terrestrial organisms, with principal application to spacecraft sterilization and, hence, planetary protection issues relating to forward contamination. Recommendations regarding specific sterilization techniques and levels of sterilization to avoid contamination of other planetary bodies go beyond the original purview of the workshop and this report. From the point of view

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques TABLE 5.1 Biosignatures: Specificity for Life Detection, and Applicability to Detecting Extant and Extinct Life and Terrestrial Contamination of Spacecraft Signatures of Life Application for Life Detection Specificity Fossil and Nonterrestrial Life Detection Morphology (Micro-and Macroscopic) Shape; size; replication structures (buds, chains of cells, septa, fruiting bodies, and spores); some biominerals; macrostructures such as biofilms and stromatolite-like structures Detect extant terrestrial life, fossils, indication of active cells; application to spacecraft contamination. Shape and size are not definitive (terrestrial life is >100 nm in diameter); replication structures are definitive indicators of life; can identify eukaryotes; biofilms and stromatolite-like structures could be definitive. Replication structures can be definitive; size, shape, and numbers of identical morphotypes such as are seen in biofilms or laminated structures observed in stromatolites may or may not be definitive for life, and additional chemical and isotopic analyses are necessary. Organic Chemistry and Biochemistry Cell walls (variety of biopolymers) Membranes (fatty acids) Nucleic acids (DNA, RNA) Proteins Hydrocarbons, steroids, hopanes Amino acids Organic metal and phosphate compounds Porphyrins, flavins, etc. Carbohydrates Nucleic acids with same genetic code as terrestrial life would likely indicate terrestrial contamination; steroids generally indicate eukaryotes; hopanes found in cyanobacteria; chirality and presence of the 20 key amino acids associated with terrestrial life indicate terrestrial and spacecraft contamination. Bacteria, archaea, and eukaryotes have specific cell-wall chemical structures; chirality, enantiomeric excess, and repeating structural units such as C5, C6 (sugars), C2 (polymethylenic lipids), C5 ( polyisoprenoids), α-substitution of protein amino acids, and L-amino acids and D-sugars are canonical for terrestrial life. Nucleic acids, proteins, and phosphates and organic-phosphate compounds could be indicative of recent or extant life. Hydrocarbons, steroids, and hopanes have been observed in the fossil record; other macromolecules (nucleic acids, proteins, and carbohydrates) are extremely labile. Nothing is known about the long-term stability of cell-wall polymers of archaea and their chemical transformations during fossilization. Inorganic Chemistry Iron minerals (e. g., magnetite) Sulfur compounds Carbonates Silicates Other biologically important metals (e.g., Cu, Mo, Ni, W, etc.) Nitrogen compounds Phosphorus compounds Ratios of biologically important elements (CHONPS) Disequilibrium in biologically important oxidation- reduction couples Best application is as additional information in conjunction with microscopic, isotopic, and organic chemical analyses for fossils and possibly for detecting presence of living extraterrestrial organisms; probably not applicable for detecting spacecraft contamination. C, N, and S can be highly specific for terrestrial life in conjunction with stable isotope or organic analyses. Some bacteria form iron compounds with highly specific structures (e.g., magnetosomes and the ferruginous ribbons formed by the bacterium Gallionella spp). Other microbes deposit silicates and carbonates and elemental sulfur as metabolites. Some crystal structures of magnetite are thought to be produced only by organisms. C, S, and N isotopes are essential additional targets of analyses for inferring past life. Oxygen isotope ratios associated with phosphates may be indicative of life. Heterogeneous distribution of biologically important minerals (Cu, Mo, Ni, W) and disequilibrium in the chemistry of rock samples could be supporting evidence for past life.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Isotopic Analyses Carbon, nitrogen, sulfur, oxygen, and possibly heavy metals Best application is to confirm extant and fossil life where there may not be evidence of intact cells; new methods also allow stable isotopic analyses of individual organic molecules and iron. Probably not applicable to detecting spacecraft contamination. Stable C, N, and S isotopes can be definitive indicators of different metabolisms. Best used to detect CO2 reduction by photosynthesis, chemosynthesis, methanogenesis, and sulfate reduction. Vital analyses to help confirm biogenic origin of minerals or cell-like structures observed microscopically on rock or soil samples. Used to understand the nature of carbon, nitrogen, and sulfur cycles in ancient environments. Environmental Measurements Global atmosphere measurements (spectral identification of volatiles such as ozone, hydrogen, methane, oxygen, water) Macroscopic life forms (imaging systems) Identify metabolic indicators of extant life, potential for habitability, and sites of high concentrations of volatiles and water; visual indication of vegetation or other indications of life. Other measurements necessary to confirm presence of living organisms and ecosystems. Not applicable for detecting extinct life unless there is some visual indication of past vegetation such as stromatolites.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques TABLE 5.2 Techniques Used to Measure Biosignatures Biosignature and Technique Measurement for Life Detection Sensitivity Limitations and Developments Needed Workshop Paper by Global Spectroscopy (TPF) Spectral lines in planetary atmospheres of extrasolar planets Oxygen: 1% of Earth atmosphere; other gases to be determined Imaging interferometry needs technical development Kasting Macroscopic imaging systems (e.g., Galileo's Solid State Imager) Morphology of macroscopic life and ecosystems Less than ~10-m spatial resolution Solar system objects only Soffen Morphological Light microscopy Structure; evidence for viability (motility, biofilms); noninvasive 0.2-µm spatial resolution Morphology only; no chemistry — Electron microscopy (environmental SEM [ESEM], SEM, EDX) High-resolution morphology and chemical composition (ESEM is noninvasive) 1-10 nm, 0.2 keV, 1% relative abundance Requires contamination-free microscopes Barker and Banfield Electron microscopy (TEM, SAED, EELS) Structure, redox state, mineralogy; invasive 1 nm, 0.2 keV, 1% relative abundance Invasive sample preparation — X- ray microscopy Electronic state of molecules Nanometer resolution Requires sectioning Jacobsen Fluorescence microscopy Structure; detect very small entities, macromolecules (nucleic acids and proteins); identify organisms (16S and 18S rRNA) and possibly viability number of ribosomes; mRNA) Can be used to enumerate viruses (30- 50 nm) Better preparation methods needed with rock and soil samples; at present time need to dislodge organisms for FISH Stahl, D. McKay, Jacobsen, Cady CT scan- XAFS imaging Internal structure; noninvasive Millimeters to centimeters Higher spatial resolution needed — Mineralogical SEM-EDX Structure and abundance of elements 1-10 nm, 0.2 keV, 1% relative abundance Reduce diameter of EDX beam (<200 nm) Barker and Banfield, Kirschvink Ion and electron microprobes Chemical and isotopic composition Single organic molecules — D. McKay Light microscopy, optical broadband spectroscopy Chemical composition; noninvasive 0.2-µm spatial resolution Limitations in spatial resolution Jacobsen, Cady, D. McKay

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Infrared (IR) spectroscopy Structure and composition 1-µm spatial resolution Improve signal- to- noise ratio — XRD-XRF X-ray structure; minerals and elements; noninvasive 200-µm spatial resolution Develop higher spatial resolution — Mossbauer spectroscopy Fe valence Bulk sample Measures Fe only — Organic Chemistry GC-MS Chemical composition, enantiomeric excess, diasteriomer specificity, structural isomer preference, and repeating structural units; lipid biomarkers; isotopes Mass resolution 1:60,000; 10–15-10–18 mole Development of ionization techniques Moldowan, Chang, Cotter, Soffen Chromatography for chirality (capillary zone electrophoresis) Structure and chirality; enantiomer excess and repeating structural units Picomoles Disadvantage is need to derivatize sample Bada Laser desorption-laser ionization (TOF, MALDI, ESI) Intact biomolecules 10–20 mole Measures molecular weight only; need to develop better lasers and improve sample preparation — Raman (IR spectroscopy and UV fluorescence) Presence of organic compounds, pigments, biomineralization; noninvasive 1 µm Need to develop low-noise detectors Becker GC-isotope ratio mass spectrometry (CHONS isotope analysis) All biogenic elements and isotopic composition Nanomoles to picomoles Improve chromatography and ion source — Chip chromatography-microarray antibody binding Single organic molecule Single molecule Need further development of sensors, detectors, and arrays — Liquid chromatography Suitable for detecting enantiomer excess, diasteriomer specificity, and repeating structural units Micromoles Improve resolution — Molecular and Biochemical Polymerase chain reaction and sequencing Detect and sequence DNA and RNA; identify specific taxa Theoretically can detect a single cell Develop methods for in vivo PCR on single cells and fossilized cells Stahl, Ruvkun et al.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Biosignature and Technique Measurement for Life Detection Sensitivity Limitations and Developments Needed Workshop Paper by Nanopores Size and some structural information about biopolymers Single molecules Nanopore technology in development phase; linear molecules only Meller and Branton, Deamer Protein- chip, chromatography, stable isotopes Molecular weight and structure of macromolecules; isotopic composition Picomoles to femtomoles Need further development of sensors and detectors Fogel Metabolic analysis Detect biological activity including metabolic pathways and bioenergetic and biocatalytic activities Not known and would be test specific Methods in the early development stage Stahl Isotopic Analyses (see above for GC- MS and IR spectroscopy) C, O, N, and S isotopes by gas source MS Isotope composition Picogram Preservation of signatures (labile compounds) — C, O, N, and S (?) isotopes by spectroscopy Isotope composition ? Development of new diode lasers and detection systems — C and S isotopes by ion probe MS Isotope composition Sub-picogram Improved detection systems and ion sources — Transition metal isotopes by MC-ICP-MS and TIMS Isotope composition Nanogram Better understanding of fractionation systematics; improved detection systems and ion sources Anbar NOTE: Acronyms used in this table are defined in Appendix A.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques of the committee the main issue regarding sterilization is the ability to sample, poststerilization, the remaining level of terrestrial microorganisms to ensure that it is below the value required for a particular mission. Because all terrestrial organisms rely on the same basic biochemistry—specifically and most importantly, the RNA and DNA nucleic acid bases—amplification to detect very small remnant levels of contamination is a suitable approach. The more difficult challenge is the sterilization itself, which must be done in such a way as to avoid damaging spacecraft components. Sterilization via dry heating in an oven, as performed on the Viking Mars landers, puts harsh demands on spacecraft components and leads to a substantial increase in mission costs and, possibly, the chances of mission failure. Sterilization by particle irradiation may not reach all spacecraft subsystems, particularly when the mission design dictates shielding electronic components from ambient sources of radiation, for example, in the Jupiter system. Irradiation levels may have to be so high and so sustained that protection of optical components, as well as electronics, becomes problematic. Radiation-tolerant bacteria may dictate that irradiation levels exceed even the extraordinary levels expected to be experienced during the prime mission phase of the Europa Orbiter.1 Finally, access to all parts of a spacecraft for sampling after sterilization is an unsolved problem. Particularly for compact landers and entry probes, access may mean disassembly and reassembly, increasing mission risk as well as the possibility of recontamination of the spacecraft. Indeed, the very compact Huygens Probe that will land on Titan in 2004 was not sterilized to a high standard on the grounds that the profoundly cold Titan environment would sterilize the lander soon after landing. Yet Titan is itself a target for investigating advanced stages of organic chemistry that on Earth might have led to life. An illustrative summary of sterilization techniques is given in Table 5.3. The choice of technique will be mission specific and may evolve, for later missions, as more is learned about the planetary target. The principal TABLE 5.3 Sterilization Techniques in Common Use Procedure—Target Technique—Problems Dry heat—exterior or interiors 105-180 °C for 1 to 300 hours—problems caused by thermomechanical incompatibility between materials can lead to the failure of electronic components, alteration of organics, and volatilization Wet heat—exterior or interiors 120-134 °C for 3 to 20 minutes—problems caused by steam (e,g., corrosion and water absorption) Alcohol wipes—exterior surfaces Isopropyl or ethyl alcohol swabbing—problems arise because interior and encased surfaces (e.g., electronic components) are inaccessible Ethylene dioxide—exterior or internal exposed surfaces Toxic gas, 40-70 °C—problems arise because the gas reaches only exposed surfaces, is absorbed by carbon polymers (e.g., rubbers and polyvinyl chloride), and leaves an organic residue Gamma radiation—exterior or subsurface Typically 2.5 Mrad—problems encountered include optical changes in glasses, damage exposed to electronics and solar cells, and altered organics Beta radiation—exterior or near-surface 1-10 MeV—problems arise because of limited penetration Hydrogen peroxide plasma—exterior or internal exposed surfaces 6 mg per liter of H2O2—problems encountered because unexposed surfaces remain untreated Ultraviolet—exterior surfaces 5,000-20,000 J/m2—problems arise because unexposed surfaces remain untreated Methyl bromide, chlorine dioxide, and ozone—exterior or internal exposed surfaces Toxic gases—problems encountered because unexposed surfaces remain untreated and because the gases may catalyze chemical reactions between metals and other components SOURCE: Space Studies Board, National Research Council, Preventing the Forward Contamination of Europa, 2000, and The Quarantine and Certification of Martian Samples, 2002, National Academy Press, Washington, D.C.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques conclusion the committee drew from the workshop and from its subsequent deliberations is that the difficult part of sterilization is not the detection of residual terrestrial contamination—it is the sterilization itself. The committee recommends that studies of future missions to astrobiologically interesting targets include explicit consideration of the types of sterilization for spacecraft systems, subsystems, and components and that sterilization costs be included in a realistic fashion. The committee recommends that special near-term emphasis be given to the issues of sample selection, spacecraft sample handling, and sample characterization. The committee also encourages further work to refine sterilization approaches to minimize impacts on mission costs and success. DETERMINING IF THERE ARE LIVING ORGANISMS IN A RETURNED SAMPLE The committee concludes that a number of very sensitive and specific techniques are available for detecting living organisms in a returned sample; however, these techniques depend on the organisms being composed of essentially terrestrial biopolymers. While other techniques exist for detecting a potentially broader suite of nonterrestrial-like (but carbon-based) organisms, their results will not be as definitive. Hence, multiple approaches will be required to establish the presence of life in a definitive fashion, unless such life happens to be essentially terrestrial in nature. There is a pressing need to develop methods for the detection in single cells of evidence of metabolic activity and of specific macromolecules, including an analysis of their chemical structure and isotopic signature. In considering the search for life on other planets, many workshop participants assumed that “life,” as defined, would be broadly like that on Earth. Because the basic functions of catalysis and replication require molecules with a high degree of specificity, life anywhere would rely on macromolecules—indeed complex polymers. Hence it would be based on carbon with its high cosmic abundance and unique propensity for building such molecules. Likewise other cosmochemically abundant elements—hydrogen, oxygen, phosphorus, nitrogen, and sulfur—should be important components of life anywhere. Much less certain is whether extraterrestrial life would use the same polymers for catalysis and replication as life on Earth—namely, proteins based on the biologically common amino acids and the RNA-DNA coupled system. Although it is possible to rationalize why terrestrial biology employs the molecules it does, much simpler life forms or those in a very different chemical environment might employ different molecules. Possibilities include a different set of amino acids, alternatives (or precursors) to RNA involving a peptide backbone, or a sugar-based nucleic acid with different bases (or number of bases). Likewise, the diversity of metabolic processes seen in the bacterial and archaebacterial domains of earthly life may not represent the full spectrum of possible metabolisms that could be encountered on other worlds. Finally, different sets of membrane lipids might be expected as well. The last is an important issue since membranes may be universal in all biochemistries and lipid biosignatures have been a key to tracing biochemical evolution in Earth's rock record. For these reasons, there is a disconnect between those techniques that have been developed to an exquisite degree of sensitivity to identify terrestrial organisms and those that could provide the greatest probability of detecting exotic life forms from another planet. Most of the chemistry-based techniques discussed in the workshop assumed terrestrial-type biochemistry or something close to it. Although techniques for amplification of nucleic acids have, for example, improved somewhat in the breadth of genetic material amplified, they are still extraordinarily narrow compared with the range of possible encoding schemes one can imagine. Imaging techniques that reveal structures indicative of processes that would be unexpected in abiotic chemistry, and whose detectable presence is independent of chemispecific amplification techniques, are promising in the sense of being quite general. These techniques are applicable to signs of extinct life as well (see below), but they may be more definitive in identifying extant life since the signatures will be much fresher than for fossils of long-extinct organisms. However, it is difficult to assess how well current experience with terrestrial biosignatures will map onto the range of morphologies or chemistries possible in extraterrestrial environments. The committee is strongly encouraged by the multidisciplinary efforts to define the possible range of processes indicative of living organisms. Given the extreme difficulty (or impossibility) of inductively describing all possible living processes based on terrestrial biochemistry, no single approach, or even

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques combination of approaches, will guarantee success on a given sample. Multiple approaches, both chemical (including isotopic and molecular) and microscopic, are key to the successful detection of life in a sample. With respect to analysis of returned samples to assess possible hazards to terrestrial life, the committee notes that there have been several recent Space Studies Board and other studies on the risks potentially posed by returned samples.2 Only insofar as the threat to the terrestrial biosphere might come from organisms whose catalytic and reproductive (information-carrying) machinery is virtually identical to our own is the reliance on amplification techniques appropriate. The continuing increase in sensitivity of amplification techniques will certainly provide increased confidence in protocols to assess the threat of forward contamination of other planets. The issue for back contamination, however, is whether organisms might exist that are sufficiently different from terrestrial organisms to escape laboratory detection, yet similar enough to pose a threat to the health of the biosphere. In the debates about life detection and back contamination, this “niche” has not been explored to the extent that it should be—in part because of the difficulties in answering the question. The committee recommends that a focused study be done in the near future to address the detection of microorganisms with varying degrees of nonterrestrial biochemistry, and the possible threat that such organisms might pose to terrestrial organisms. There can be no single strategy for sample acquisition since each planetary body presents a unique contemporary geology or chemistry and a unique evolutionary history. Similarly, there can be no single strategy for treatment of returned samples. Instead, the risk factor related to each variety of sample must be assessed, and our experience with lunar return samples can serve as a useful guide in developing policy. To the extent possible, reasonable efforts (defined through carefully deliberated scientific strategies) should be made to assess the potential for extant life on other planetary surfaces in situ, using robotic missions. The results will markedly increase confidence about the risk factors associated with a given sample that could be returned to Earth for further study and will provide scientific evidence to further justify the expense of a return mission. Since life (or past life) will concentrate in habitats that provide suitable nutrients and chemical or physical conditions, its distribution on any planetary body will be patchy and of varying local abundance. For Mars in particular, the issue of selecting promising sites has been addressed in multiple reports and studies, the most recent being that of COMPLEX.3 At the same time, in situ analysis of a site will be limited in the capability and flexibility of the experiments that can be employed, compared to those that can be brought to bear on returned samples. The potential phase space of possible life forms and biomarkers is broad, and in situ studies are more likely to miss detection than are studies performed on returned samples. Nonetheless, the combination of site selection and analysis followed by acquisition of the most promising samples for study on Earth is likely to maximize the chances of success in the identification of extant or extinct life. Appropriate site selection for sample return is critical and will determine the amount of sample required for testing and the need for possible sample concentration. Multiple measurements with different techniques will be required to perform triage on a set of field samples at a given landing site, so as to select the most promising samples for in situ or returned life detection. Sampling and return methods must be compatible with the requirements imposed by analysis. For example, an activity-based measure (metabolism) will require that samples not be exposed to environmental extremes during recovery and return. A (bio)chemical assessment of structures characteristic of life (e.g., enantiomers) would not place the same constraints on sampling and return. Sample selection, handling, and characterization at a given site must be thought through carefully to ensure an interpretable result in either laboratory or in situ life detection protocols. The committee recommends that special near-term emphasis be given to the issues of sample selection, spacecraft sample handling, and sample characterization. DETERMINING IF LIVING ORGANISMS HAVE BEEN PRESENT AT SOME EARLIER EPOCH AND HAVE LEFT FOSSIL REMNANTS BEHIND IN A RETURNED SAMPLE The committee concludes that the search strategy for evidence of extinct life must include the identification of suitable landing sites, the selection of the appropriate rock types, and multiple analytical techniques that, in the aggregate, are capable of distinguishing between abiogenic and biogenic signatures. The

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques assessment of extinct biosignatures will likely require a sample return mission to carry out the sophisticated set of measurements needed to make this determination. Most of the more general techniques for detecting life, versus the very specific (and potentially more sensitive) approaches of amplifying nucleic acid bases, would be suitable for searching for either extant or extinct life. This is an important point since the majority (but not exclusive) view is that for Mars, the probability is much higher of detecting extinct rather than extant life. Biosignatures may be morphological, chemical, or isotopic. For example, the biological fixation of carbon dioxide into organic carbon preferentially leaves behind the heavier isotope, 13C. Hence, the lower value of 13C/12C seen in organic carbon compared to carbonates over geologic time testifies to the lengthy history of life on Earth (3.9 billion years or longer). This seems a very reliable signature of biological processes past or present. However, work remains to be done in fully quantifying the variation of isotopic fractionation in terrestrial microorganisms, and indeed the variation in the isotopic ratio in biologically fixed carbon is large. Also, the preservation of organic carbon in the martian soil is questionable. Identification of isotopic biosignatures in elements more robust against alteration, such as iron, is promising, but more measurements are needed to establish fractionation patterns and decide whether the approach is reliably diagnostic of past biological activity. Morphological signatures, such as mineralogical or textural alteration of minerals by organisms, can reveal the past presence of life when carefully documented and compared to known alteration signatures in the terrestrial rock record. Elimination of false positives in a sample will require multiple types of biosignatures and critical testing of alternatives to the biological hypothesis. The committee particularly commends the work to characterize biosignatures associated with microbe-mineral interfaces on Earth and strongly encourages additional efforts in this regard, especially in extreme environments. Biosignatures degrade progressively over time. Rates of degradation for a particular kind of biosignature generally depend on the environment; for example, the rates of racemization of many chiral amino acids are accelerated by the presence of water. A key issue then, and one for which much work remains to be done, is to understand the rates and nature of degradation of biosignatures in planetary environments that are likely candidates to be searched. Additional effort on basic mechanisms can be undertaken today and is largely independent of the uncertainties associated with specific, rapidly changing plans (and budgets) for obtaining samples. Application to a particular planetary sample will depend on characterizing, to the extent possible, the environmental conditions to which the biosignatures were exposed over long periods of time. The committee recommends that attention be given to understanding thoroughly the rates and nature of degradation of biosignatures in planetary environments. Theoretical and experimental studies should be supplemented with comparative analysis of putative samples of extraterrestrial biomarkers (e.g., ALH84001), with a specific eye to better understanding the issue of degradation of signatures of past life. Additionally, the identification and development of new and possibly universal biosignature approaches should be an active area of study. In the course of the workshop, an issue arose that was not part of the original charter—namely, the availability of the same samples to multiple research groups. The resolution of at least one controversy regarding the interpretation of biosignatures in the SNC meteorite ALH84001 has been hampered by the fact that distinct research groups are analyzing different samples from the meteorite, with evident sample-to-sample variations. Magnetite crystals left behind by bacteria exhibit a combination of properties that are considered indicative of biological manufacture because these properties are not found in abiotically produced crystals (either in nature or in the laboratory). As carefully studied as this phenomenon has been, its one application to an extraterrestrial sample—ALH84001—has led to a broad range of interpretation of the magnetite found therein. Multiple workshop participants held the view that the current mechanism for dissemination of samples and the normal handling practices within research groups have discouraged direct analysis of the same sample by multiple teams. Yet the scientific method encourages, if not demands, that a controversial result obtained on a sample be validated through additional studies, including by alternative research teams with different approaches. The challenge of having multiple research groups analyze the same sample will be greater for samples returned from extraterrestrial bodies by spacecraft (as opposed to serendipitous meteorite infall), since quarantine could discourage the transfer of samples from one laboratory to another. Nonetheless, the nature of some of the most vigorous workshop debates illustrated the importance of having groups with differing approaches or

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques predisposed biases examining the very same material. Possible solutions include formation of consortia of researchers from different institutions to perform a particular type of analysis, provision for multi-institutional (repeat) analyses of a select subset of the sample material, and so on. Because of these concerns, the committee recommends that any plans for analysis of returned extraterrestrial samples include a provision for repeat analyses of a subset of the same material, preferably by different teams. The committee encourages early development and testing of appropriate protocols using existing samples of high astrobiological interest (e.g., ALH84001). DETERMINING IF THERE ARE LIVING ORGANISMS OR FOSSILS IN SAMPLES EXAMINED ROBOTICALLY ON ANOTHER SOLAR SYSTEM BODY Because of the technical difficulties associated with returning samples to Earth (and potential back-contamination threats), much of the search for life elsewhere may initially be done in situ. It was evident from the workshop presentations that many of the powerful and sensitive techniques for detecting life in the laboratory are not yet available in the form of miniaturizable spacecraft instrumentation and may not be so in the near future. Consequently, in situ life detection approaches are constructed based on a priori hypotheses as to the structural, metabolic, or replicative nature of the organisms searched for. This allows a small, flyable package to be designed —but severely narrows the phase space of possible successful searches and limits the opportunity for adaptively altering the analysis of samples based on initial results. Because of the continuing rapid improvements in technology, it is not appropriate at this time to recommend a specific set of techniques for in situ life detection, but in situ life detection will require commitment to a small set of potential techniques with significant lead time to ensure that they can be space qualified. Some of the approaches available for the detection of living organisms are available in miniaturized form and are potentially space qualifiable for an in situ life detection mission. The committee encourages continued efforts to develop innovative and miniaturizable techniques for in situ life detection. It remains unclear as to which environments in our solar system should be searched for signs of life, beyond the general identification of planetary targets (e.g., Mars, Europa, and Titan). In large measure, we do not yet know enough about these bodies to target searches in particular locations. Where might organic molecules be present or have been present within the martian crust? Are there places on the surface of Europa where the putative ocean is accessible to drilling or where the extrusion of oceanic material is continuing today? Where are the interesting organics (liquid or solid) on the surface of Titan? Answering these questions requires additional planetary missions to deepen our understanding of these environments, and there is a natural tension between systematically planning and executing those missions versus pressing on with the search for life itself. The key to success in the search for life is appropriate site selection. Promising surface or subsurface sites would be ones showing evidence of past or present liquid water, aqueous alteration of minerals, sources of chemical free energy (past or present), and possibly organic molecules.4 However, terrestrial experience shows that extant life will not be confined to sedimentary environments, and biosignatures need not (indeed probably will not) consist of organic matter. Almost certainly, the most interesting sites from the point of view of the search for life will not be the easiest to get to. For Mars, this means that landing site selection cannot be based primarily on issues of spacecraft safety. Furthermore, proper site selection will require a series of missions including orbital reconnaissance followed by exploration of selected sites by landed vehicles. The in situ search for life can serve as a selective precursor for determining which samples should be returned for laboratory analysis, and assessing the potential hazard of back contamination before a sample is brought back to Earth. Site characterization (age, exposure to liquid water, high temperatures, etc.) will be essential as well in order to understand the extent to which degradation of biosignatures, analyzed in situ or on Earth, has occurred. In the case of Europa, a follow-on to the Galileo mission (most plausibly in the form of an orbiter) is required to identify potential landing sites where access to subsurface liquid water is possible. Finally, for Titan, the Cassini-Huygens mission will provide the necessary data to map the surface distribution of organics and identify whether any particular sites are of interest for future studies of abiotic organic evolution.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Living organisms represent a subset of the organic molecules that exist in the solar system and beyond. Analyses of meteorites, the atmospheres of the giant planets and Titan, and the surfaces of outer solar system bodies reveal a range of abiotic chemical processing of organic molecules. Detection of simple organic molecules (e.g., methane) has been accomplished for the atmospheres of very cool brown dwarf stars, an important precursor to a protocol for the eventual remote spectroscopic assessment of the habitability of extrasolar planets. Although the vast bulk of the analysis performed to date on extraterrestrial samples demonstrates the non-living origin of these organics, the small enantiomeric excess in the Murchison meteorite remains a contentious puzzle. Whether it suggests the action of abiotic processes to introduce asymmetry into an abiotic stereochemical system, or some sort of postimpact terrestrial process acting on the meteoritic organics, is unresolved. In the case of Mars, the failure of the Viking landers to detect organic molecules was key to the conclusion that the Viking life detection experiments were seeing abiotic processes in a highly oxidizing soil, rather than metabolism. Terrestrial laboratories are capable of detecting and characterizing very small amounts of organic molecules in samples, as well as reliably determining whether the molecules are indigenous to the sample or a terrestrial contaminant. This was of particular value in analyzing the SNC meteorite ALH84001, in which it was found that most of the organic material is a terrestrial contaminant and the remainder resembles primitive meteoritic or interstellar material. The development of more sensitive techniques to detect organic molecules and characterize the extent to which organic chemical systems may be evolving toward life in a planetary environment is an important priority. Many organic-rich environments in the solar system will be accessible to in situ analysis in the coming couple of decades but are prohibitively difficult targets for sample return (e.g., the surface of Titan). It is therefore desirable to miniaturize the instruments necessary for sensitively detecting and characterizing organic phases so that they can be accommodated on spacecraft dispatched to a variety of solar system targets. The committee concludes that it is crucial to continue the development of techniques to detect and analyze in situ organic chemical systems of either biotic or abiotic origin, with the goal of increasing the techniques' sensitivity and diagnostic capability. REFERENCES 1. Space Studies Board, National Research Council, Preventing the Forward Contamination of Europa, National Academy Press, Washington, D.C., 2000. 2. See, for example, Space Studies Board, National Research Council, The Quarantine and Certification of Martian Samples, National Academy Press, Washington, D.C., 2002. 3. Space Studies Board, National Research Council, Assessment of Mars Science and Mission Priorities [prepublication text], National Academy Press, Washington, D.C., 2001. 4. M.H. Carr in Water on Mars, Oxford University Press, New York, 1996, pp. 180-183.