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APPENDIX I INSTRUMENTATION FOR THE DETECTION OF EXTRATERRESTRIAL LIFE CARL W. BRUCH INTRODUCTORY NOTE This paper contains a review of the characteristics and state of develop- ment of some of the instruments that have been devised under the auspices of the National Aeronautics and Space Administration. The information given here is excerpted from a report prepared by the author for the present study. The instruments and experimental methods are reviewed in terms of the kinds of evidence of biological significance that they are intended to provide. MORPHOLOGICAL EVIDENCE One of the best ways to obtain evidence of macroscopic organisms or aggregations of microscopic forms is by means of a high-resolution television system. Such a device would enable us to view the surface with a resolution of 0.4 millimeters from a height of 4 meters with both wide and narrow angle fields between 25 and 45 degrees. Such an instrument is not now under development for biological purposes, but a related device has been considered for use in the program of lunar studies with the Surveyor space- craft. Such a television experiment has limitations in that it requires large amounts of power and very considerable telemetry facilities. 487

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488 APPENDIX I Microscopes Development of a vidicon microscope for detecting extraterrestrial life, suggested by Joshua Lederberg, is being carried out at the Jet Propulsion Laboratory of the California Institute of Technology. The simplest model of such a microscope for space use is called the "abbreviated vidicon microscope." It is a fixed-focus, impaction, phase- contrast instrument. An aerosol sample is injected into the focal plane of the microscope through an orifice in the condenser lens. The lens system observes a 100 micron field with 0.5 micron resolution. The vidicon picture can be transmitted in digital form with eight levels of gray shading and 400 lines per frame. Transmission of this amount of data normally would require more than 500,000 bits per frame. A second type of instrument, also under development, is the automated scanning, flying-spot, photometric microscope. This device has been used by the U. S. Army Biological Laboratories to distinguish viable organisms from dust particles in aerosol clouds. The principle of operation is as follows: particles are impacted on a clear plastic tape, which then passes through a staining solution; organic particles are differentially stained; and the tape, with adhering organisms, is passed under the flying-spot scanning objective, which monitors the opaque particles against the stained particles. A readout is obtained in terms of "dust versus organisms". The power requirements of this instrument would be much smaller than those of the vidicon microscope. Both of these instruments have been brought in development to the stage of experimental breadboard models. CHEMICAL EVIDENCE Gas Chromatograph Several chemical detectors for biological compounds are under develop- ment. The first of these, a gas chromatograph, is under development also at the Jet Propulsion Laboratory, with the assistance of Sanford Lipsky of Yale University and James Lovelock of Houston University. The basic principle by which the experiment works is the qualitative and quantitative separation and identification of pyrolytic products of organic compounds found in biological systems. This system is based on gas-liquid chromatog- raphy, which separates compounds by their variance in distribution coeffi- cients and detects them by means of several sensitive devices. The present experiment is designed to reveal the presence of organic matter in soil

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Instrumentation for the Detection of Extraterrestrial Life 489 samples obtained from the Martian surface. It can suggest the presence of life by producing fingerprint chromatograms of the pyrolytic products of known biochemical compounds, living organisms, their metabolic products, or the degraded products of dead organisms. Preliminary work on the gas chromatography of the volatile products from pyrolysis of microorganisms suggests that fingerprint chromatograms can be obtained that will identify proteins, lipids, carbohydrates, possibly nucleic acids, and other biochemi- cal compounds. If chromatograms are obtained that are not indicative of any pretested terrestrial materials, interpretation of the data would be made by attempting to duplicate the chromatograms in the terrestrial laboratory under conditions identical to those existing during the performance of the experiment on Mars. Furthermore, preliminary data show that high sensi- tivity optical resolution of diastereoisomers of neutral amino acids can be achieved by gas chromatography. Controls include calibration samples of known organic materials that can be introduced into the instrument just prior to the performance of the experiment. Functional and environmental parameters will be monitored to verify the conditions of the experiment. The sensitivity of this device is in the range of 0.1 to 10 micrograms of organic sample and a single sample that can be acquired by a roving vacuum aerosolizer is adequate. In operation, the crude sample is sub- jected to pyrolysis and the volatile degradation products are introduced into the separation column of the chromatograph. The components of the instrument include a carrier gas storage tank, gas pressure regulator, sepa- ration column, ovens, ionization detector, and electronic circuit compo- nents. All instrumentation requirements are satisfiable within the present state-of-the-art; but some of the components need further development in order to be able to survive flight conditions. The present status of the experiment is somewhere between a functional- feasibility breadboard and a flight-sized breadboard. A flight-sized instru- ment is estimated to weigh about 7 pounds, occupy a volume of 200 cubic inches (without apparatus for sample acquisition), and require 4l/2 watts of power. Data are taken for a 40 minute period and require special treat- ment in storage and transmission. Orientation of the instrument on the surface of Mars is not a crucial consideration, but the instrument must be protected against the temperature of the Martian night. It will survive within the temperature range of —50°C to -)-135°C. Radiation may affect the detector but it is small enough to be shielded. No pressure effects are anticipated. It is believed that the instrument can be sterilized by dry heat, but the effects of this treatment have not been evaluated. Surface sterilization can be obtained with ethylene oxide.

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490 APPENDIX I Mass Spectrometer A second instrument for deriving chemical evidence of the presence of extraterrestrial life is the mass spectrometer being developed by Klaus Biemann at the Massachusetts Institute of Technology. At the present time the experiment is in the conceptual state. Standard mass spectrometers are being used to examine synthetic mixtures of organic compounds. The mass spectra of amino acids, peptides, nucleosides and carbohydrates have been investigated in detail to evaluate the potentiality of mass spectrometric techniques for the identification and structure deter- mination of organic compounds of possible extraterrestrial origin. Com- plete groups of compounds obtained by breaking down an entire living organism have not yet been examined. No reference controls are required except for machine operation. The sensitivity is in the range of 1 microgram to 1 nanogram. The projected miniaturized mass spectrometer will have a range of 0 to 250 mass units and may be operated in the range of 0 to 160 mass units. Only one sample is required. To date, no sampling technique has been devised. The sample must be ground, placed on the tube filament, degassed and heated by the filament. The tube must be evacuated after the sample has been inserted. The complete instrument includes a miniaturized mass spectrometer, vacuum pump, sampling device, electronics, and an analog-to-digital con- verter. The instrumentation is within the present state-of-the-art. Based on current estimates, the flight instrument (exclusive of sample collection equipment) would weigh about 15 pounds and would occupy about 400 cubic inches. The power requirement is 15 watts, and the number of bits is between 5,000 and 10,000. A meaningful spectrogram involves the taking of at least 250 pieces of information. The instrument would not require any special protection from the space environment other than that normally required for electronic instrumenta- tion. Temperature, radiation, impact and vibration have no adverse effects. There is serious doubt that the electronic components of this device will stand the presently recommended dry heat sterilization cycle of 135°C for 24 hours. The unit could be surface-sterilized with ethylene oxide. It may be possible to assemble the instrument under sterile conditions, but the interiors of the components would still be contaminated. It must be pointed out that one of the most serious problems in the development of this instrument is that of sample acquisition, which has not been here considered in any detail.

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491 Gas Chromatograph with Mass Spectrometer A third way of obtaining chemical evidence of extraterrestrial life is the combination of the gas chromatograph with the mass spectrometer. Both instruments have been proposed as broad spectrum analyzers for planetary atmospheres and for the analysis of volatile or pyrolizable materials in planetary and lunar soils. Each instrument has inherent strengths and weaknesses, and each is a powerful analytical device in its own right. Recently, a technique has been developed that permits the combination of these two instruments into a package far more powerful than either instrument alone. The mass spectrometer provides for most molecules an unequivocal determination of molecular weight and fragmentation products. For most relatively simple mixtures the mass spectrum provides quantitative de- termination of each component. However, there are many complications in the interpretation of mass spectra. One example of such a complication is the problem of mass doublets. Components such as ethylene, nitrogen, and carbon dioxide, each having a mass to charge ratio of 28, fall on top of each other in portions of their spectra. If large enough amounts of each component are present, a rough quantitative estimation of each may be made on the basis of fragmentation products, but this technique is difficult and inexact. Another complication arises in the analysis of high molecular weight organic compounds of biochemical interest. In such compounds the probability of fragmentation is much greater than the probability of ionization of whole molecules; thus, many different compounds give the same fragmentation products and make the resolution of mixtures extremely difficult. The gas chromatograph in general separates complex mixtures cleanly into individual components. The identification of the individual components is usually inferred from their retention time in the column. This technique is suitable for laboratory application given some a priori knowledge of the input sample and given the availability of each component in the pure state for calibration. For extraterrestrial application it is at once apparent that identification by retention time is much less positive, and the probability of obtaining a completely unknown and unidentifiable constituent in the sample is quite high. The technique of separating complex mixtures by gas chromatography and analyzing the effluent stream by mass spectrometry has been applied in many laboratories here and abroad. The principle difficulty in this tech- nique is that a large volume of carrier gas must be introduced into the mass spectrometer along with the sample. Extreme sensitivity is required, since the mass spectrometer must be held at pressures on the order of 10"5 torr

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492 APPENDIX I and the material of interest comprises only a very small fraction of this total. Within recent months a technique has been developed for the separation of the sample from the carrier gas by use of a pseudo-molecular beam device. Advantage is taken of the momentum of the heavier mole- cules flowing from the column to enhance their concentration by differential pumping to a level at least a hundred higher than in the original mixture. Submicrogram samples have been completely resolved and analyzed. Development of instrumentation of this sort will be carried out at the Jet Propulsion Laboratory with the assistance of S. R. Lipsky of Yale Uni- versity and K. Biemann of MIT. It is anticipated that an instrument pack- age capable of atmospheric analysis during parachute descent and subse- quent analysis of the pyrolytic products of surface materials may result from this developmental program. Optical Shifts in Dye Complexes A fourth approach of obtaining chemical evidence of extraterrestrial life involves an experiment called the "J-Band" and is being developed by R. E. Kay of the Aeronutronic Division of the Philco Corporation. It is intended, by visible spectrometry, to detect the shift of the absorption band of a dye (thiocarbocyanine) to one or two new wavelengths upon the interaction of the dye with certain organic macromolecules such as proteins and nucleic acids. As with most dye complexes, a high salt concentration can obviate the results. The use of a pyrolyzed or oxidized soil sample extract is required as a control in the reference cell. Reference is obtained by dye samples in solution in both a test and a reference optical beam to determine a zero null. Alternately, a standard such as polyglutamic acid could be employed at the conclusion of the determination. The sensitivity is in the range of one gamma of biologically important macromolecules. A single sample of soil which is processed by extraction, drying, and either pyrolysis or dialysis followed by a conductivity check is required. The sample acquisition mechanism needs much further development. One complete cycle is a minimum; however, recycling is planned to be internal to the experiment with the possibility of using two different temperatures. At the present time the experiment has been demonstrated only under labo- ratory conditions. No breadboard equipment has been constructed. The instrumentation required is a double-beam, optical-null spectrophotometer together with a light source, detector, amplifiers, and a sample processor. It is estimated that an appropriate instrument would weigh at least 5 pounds, use 3 watts of power, and have a data bit requirement of about

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Life Detecting Instruments 493 500. It would need protection against the low temperatures of the Martian night. The ability of this instrument to tolerate the presently recommended heat sterilization cycle is unknown. The dye will have to be sterilized by a "cold" technique. Sterile assembly does not appear to be applicable to this instrument. The main problem area in the laboratory feasibility study is to have more testing to determine finer distinction between classes of macromolecules and among specific molecules within a class. Ultraviolet Spectroscopy Detection of the absorptions near 1800A that are due to peptide linkages has been proposed as a method for obtaining chemical evidence of the presence of living material. Experiments have been carried out on a wide variety of amino acids, dipeptides, tripeptides, polypeptides, and proteins. It was found that all substances containing peptide bonds exhibited an absorption maximum in the 185-190 millimicron region. Experiments with substances that might give false positive absorption showed that many non-peptides similarly absorbed in this region. However, it was observed that hydrolysis of the peptides resulted in a decrease in absorbancy, as did hydrolysis of the extracts of soil and sand. This effect might distinguish between peptides and non-peptides. However, a recent investigation by R. D. Johnson of the Ames Research Center showed that interference from non-peptide materials coupled with stray light errors would seriously compromise this approach to life detection. Interest is now centered in the possible incorporation of a refinement of this technique with the optical rotatory dispersion experiment described below. Optical Activity The sixth, and probably the most important, of the chemical approaches to detection of extraterrestrial life is that of optical rotatory activity. An instrument for this purpose is under development by Ira Blei and John Liskowitz of Melpar, Inc. It is designed to measure the optical rotation of plane polarized monochromatic light at 240-260 millimicrons when it is passed through a solution prepared from a soil sample. Depending on the nature of the extraction procedure, the source of such optical rotation would be either proteins, nucleic acids, or nucleotides produced as a result of biological asymmetric synthesis. The control required for this experiment is a quartz plate of known

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494 APPENDIX I optical rotation that will be introduced into the light path. This calibration will serve to establish a base line as well as to establish the functioning of the instrument. The sensitivity of this device, in terms of chemical com- pounds, is in the region of 1-10 micrograms. In terms of biological organisms, approximately 104 to 10° organisms would be required for a positive response. No sampling method has yet been developed for this device. One sample per analysis is required. After collection, the sample will be extracted with an alkaline solution (probably sodium hydroxide); following extraction the solution would be clarified and then introduced into the chamber for observation. The instrumentation is within the present state-of-the-art and it is esti- mated that a flight-sized instrument would weigh about 6 pounds and occupy a volume of 130 cubic inches. The power required is 2 watts, and the number of bits to be transmitted is approximately 20. The instrument is extremely durable and would need no protection from low temperatures. There is doubt that the electronics of this device will tolerate the presently recommended dry heat sterilization cycle. Ethylene oxide can be used for surface sterilization. An instrument with sterile surfaces could also be achieved by means of sterile assembly. The instrument is, at present, in the stage of engineering breadboard design. PHYSIOLOGICAL EVIDENCE Several instruments that depend on the detection of metabolic activities, growth, and reproduction are under development. These and other investigations that may lead to new instrumental designs are described below. "Multivator" This apparatus was devised by Joshua Lederberg of Stanford University, and is under development there. The principle experiment to be carried out with this device is the determination of the presence of specific catalysts, particularly phosphatase. However, the Multivator is a biochemical probe that could conduct a variety of biochemical or biological experiments on Mars. The variety of these experiments is limited only by those biological properties that can be measured by a photomultiplier tube as an output transducer. The device can be described as a group of cells or tubes in which samples are introduced and combined with appropriate reagents or

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Life Detecting Instruments 495 biological materials. The resulting reactions are then detected with a photomultiplier. It has also been considered for use in the detection of biologically important macromolecules by fluorimetry, turbidimetry, nephelometry, absorption spectroscopy, or absorption spectral shifting in a test substrate. The basic elements of the instrument are a light source, followed by a filter, the sample under investigation, another filter centered at either the same wavelength as the excitation filter for colorimetry or light scattering, or at a different wavelength if fluorimetric observations are to be made, and finally, a light detector, usually a photomultiplier. The most recent version of the Multivator consists of 15 modules ranged in a circle with an impeller in the center of the circle. Each of the modules comprises a reaction chamber, solvent storage chamber, tapered valve pin, explosive charge bellows motor, and a filtered light source. The entire solvent chamber is sealed prior to operation by means of a thin diaphragm placed in front of the pointed valve tip. In operation, dust-bearing air is drawn through the impeller and in front of the reaction chambers. The impeller imparts sufficient velocity to particles larger than 10 microns in diameter to fling them into the reaction chambers, where they tend to settle. Upon completion of the particle- collecting operation, the bellows are operated electrically. Expansion of the bellows results in sealing of the reaction chambers and injection of the solvent. The substrate materials, which have been stored dry during flight in the reaction chambers, are dissolved and the reaction begins. After a pre-set reaction time, the excitation lamps are turned on sequentially and the light signal, or fluorescence level in the case of the phosphatase assay, is detected by the photomultiplier. This information is then reduced to digital form and transmitted. The only control required is a reference cell without a soil sample. Actually, the device contains three dummy chambers for control purposes. The biological sensitivity of the present apparatus in the phosphatase assay is approximately 105 organisms per milliliter, and the chemical sensitivity is in the microgram range. Live organisms are not required for this experiment. At present the sample is obtained through the action of the impeller, which acts as a sort of vacuum cleaner. With sample collection included, present estimates indicate a weight of about 3 pounds; a volume of about 90 cubic inches; and a power require- ment of a maximum of 5 watts. The number of data bits required for this experiment is approximately 5,000. The present device is not affected by radiation, pressure, impact or vibration; but the temperature must exceed 0°C for the experiment to proceed. Sterilization of the instrument is planned as a two-stage operation. First the Multivator, less substrate, will be sterilized by heating to 135°C for at I

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496 APPENDIX I least 24 hours. The sterile substrates are introduced into the instrument in a sterile glove box. It is possible that the instrument can be surface- sterilized with ethylene oxide gas. It is not known whether or not the instrument could be assembled under sterile conditions. In the opinion of the experimenter, this device is almost ready for flight hardware development. However, a stable organic reagent for use in the phosphatase assay remains to be demonstrated. From this point of view the Multivator is still in the conceptual stage, and no hardware develop- ment will be undertaken until satisfactory dye reagents are developed for the phosphatase assay. "Gulliver" Gilbert Levin of Hazelton Laboratories and Norman Horowitz of the California Institute of Technology have designed an apparatus known as Gulliver. It is intended to detect bacterial growth by determining the formation of radioactive CO2 from C14 labelled substrates. The present medium consists of basal salts fortified with soil extract and containing the organic C14 substrates as formate, glucose, lactate, and glycine. If the experiment were to function as planned, it would indicate the presence of a biota that could utilize the added sources of carbon and nitrogen for growth. If the production of carbon dioxide were exponential, as is charac- teristic of binary fission, growth would be indicated. If the production were not exponential, it would not be clear whether the carbon dioxide release were the result of exogenous enzymatic degradative metabolism in the absence of growth or chemical degradation of the radioactive substrate. The success of the experiment depends on being able to supply the appropriate environment for growth. The use of controls is also difficult because identical samples are not provided to both the test and control chambers. During present field tests, an inhibitor of bacterial growth is introduced into the growth chamber of one of the two units. This addition should result in a cessation of CUO2 formation. The number of organisms required to give a positive response has varied during these field trials; but it appears that the soil should contain approximately 10,000 organisms per gram if a positive response is to be obtained. A single untreated sample is required. The sample is acquired on a length of chenille attached to the end of a line, which is ejected from the instrument. The line is dragged across the surface to collect soil particles, and these are introduced with the line and chenille into the growth chamber. Recent estimates show a weight of 7 to 12 pounds; volume between 300 and 600 cubic inches; and power requirements of 3 watts average, and 5 watts peak, without heater (an additional 2l/2. watts is estimated for a

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Life Detecting Instruments 497 heater). The total amount of data for this experiment is 700 bits. The apparatus consists of a sample collecting mechanism, a main chamber containing a sample processing mechanism, a radiation counter, an electronic signal and data processing system, an electronic programmer, and a heater. All of these items are within the state-of-the-art. The instru- ment is not affected by temperature, radiation, pressure, impact or vibra- tion. Incubation temperature will have to be kept above the freezing point of water. It is not known whether the electronics of this system will stand a heat cycle of 135°C for 24 hours. The designers indicate that this heat cycle should present no serious problems. They also state that the instrument is suitable for sterile assembly. At the present time the experiment can be considered in the status of an advanced flight-sized breadboard model. This experiment appears to be the farthest advanced in terms of engineering of all those now under development. "Wolf Trap" Measurement of microbiological growth is the functional basis of an instrument known as the Wolf Trap. The experimenters are Wolf Vishniac and C. R. Weston of the University of Rochester, and development is being carried out by Ball Brothers Corporation. The experiment is designed to detect the growth of organisms by light scattering measurements and by change of pH. The relative concentration of microorganisms in a turbid culture can be estimated by measuring either the attenuation of a beam of light passing through the suspension, or the intensity of the light scattered by the organisms. The measurement of the scattered light (nephelometry) has been selected because of its inherently greater sensitivity. The measurement of turbidity and pH has some drawbacks. If the sample introduced into the device contains large amounts of colloidal materials, the background scattering might obscure any changes in turbidity due to microbial growth. Furthermore, the presence of hydrophilic sub- stances, which can swell, could produce light scattering without growth. If the sample contained highly buffered soil, changes in pH could be pre- vented; conversely, the slow release of relatively insoluble acidic or basic substance would give a change in pH without growth. With the present optical system in the two chambers of the engineering breadboard model, the Wolf Trap gives a reliable signal at approximately 103 bacteria per milliliter. Two types of controls are planned: an inoculated blank (i.e., a non-

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498 APPENDIX I nutrient medium, perhaps only water) and an inoculated replicate to which has been added some germicidal agent, such as formaldehyde. The present two-chambered engineering breadboard of the Wolf Trap has been designed to be fully automatic, with the option of operating certain functions manually. The operation of the apparatus is controlled from a portable, battery-operated console that simulates the parent spacecraft. Upon command from the console, the sample pickup begins to operate, the collection nozzle is ejected and the gas flow initiated. Dust particles in the vicinity of the nozzle are sucked into the return line by a pressure drop across a venturi and carried by the gas into the culture chamber, where they are deposited. The pressure drop across the nozzle is adjusted, for test purposes, to a level that could be achieved at a 50 millibar Martian atmosphere. (Specifications for the present model were drawn up before it was known that the Martian atmosphere pressure was much lower than 50 mb). Trials have indicated that the present pickup design would, at best, operate marginally at a pressure of 10 millibars. Sample acquisition has turned out to be one of the most difficult aspects of the problem of designing a life detection probe. One sample is required for each chamber. Thirteen chambers, including controls, but not allowing for replication, are anticipated in the final model. The instrumentation will include a gas pressure supply, a flexible tube with an enclosure shroud to be dropped to the surface, and a venturi nozzle which will provide a differential pressure. A five-chamber system is estimated to weigh 5 pounds. The volume for five cells is about 200 cubic inches. Total power consumption is approxi- mately 1 watt. The number of data bits is approximately 300. The safe limits for experiments are the following: 1. Temperature: —50°F to 310°F. (Incubation chambers at 70-80°F.) 2. Radiation: total flux density of 1 X 107 roentgens. 3. Pressure: vacuum to several atmospheres. 4. Impact: the source lamp will limit shock loading to 2,000 g at 5 milliseconds. 5. Vibration: the unit will be designed to withstand the vibration normally encountered when boosters such as Atlas or Thor-Delta are used. The sterilization requirement has already been met in the breadboard model; it can be repeatedly heated to 145 °C for 24-hour cycles. The need for ethylene oxide sterilization is not anticipated. The unit is suitable for sterile assembly, but this method is rejected by the experimenter.

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Life Detecting Instruments 499 Development and construction of the breadboard model has been com- pleted. Detection of Photoautotrophy Preliminary work has been done at the Hazelton Laboratories in an attempt to convert Gulliver to a device which can detect the presence of a photosynthetic organism. Experiments with Chlorella pyrenoidosa have indicated the feasibility of using Gulliver to detect photosynthetic metabolic responses. A urea salts medium containing DL-sodium lactate-1-C14 was found to be a suitable culture medium. Initial responses to light change were more pronounced when the inoculated chambers were started in the dark. A Mark III Gulliver has been modified for detection of photosyn- thesis by placing the light source directly in the culturing chamber. The unit is now being tested. Detection of ATP An experiment designed to detect microorganisms by determining the presence of adenosine triphosphate (ATP) is also under development at the Hazelton Laboratories. This development is being managed by the biological group at the Goddard Space Flight Center, who are interested in determining the vertical extent of the terrestrial biosphere. This system responds in fractions of a second to the introduction of organisms. There is no requirement for growth, nor is it required that the organisms be intact or alive. The ATP-luciferin system is well suited for use as a "real-time" life detection system in biological studies of the stratosphere. In terms of planetary exploration, this experiment may not require a landing vehicle since, potentially, it can be used on a probe if a high volume sampling of the planetary atmosphere can take place during descent of the spacecraft. The reagents for this system contain luciferin, luciferase, magnesium ions and oxygen. The introduction of ATP brings about the emission of visible light. This system, in its present form, will detect approximately 10~4 micrograms of ATP. This amount of ATP can be derived from 4,000 yeast cells, 5,000 algae, or approximately 100,000 bacteria. It is hoped to increase the sensitivity by three or four orders of magnitude by use of cryogenics and synchronous detection. The control for this experiment is an uninoculated reagent mixture. As indicated above, the sample can be obtained by high volume sampling of the atmosphere, or the sticky string device used in the Gulliver can be employed. The sample can be treated with methanol or another solvent to

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500 APPENDIX I extract the intracellular ATP. A one- or two-minute extraction would probably increase the sensitivity. The instrumentation would consist of a sample collection device, an extraction device, a processing device, and a photomultiplier tube. These components are commercially available. The power and bit requirements are undefined at present, but they should be in the same range as those of the Gulliver and Wolf Trap. The instrument would tolerate the Martian environment, but the enzymatic reaction would function slowly at freezing temperatures. The instrument could probably be sterilized with the recommended dry heat cycle of 135°C for 24 hours, but the reagents would have to be sterilized by filtration. This experiment is the subject of a laboratory feasibility study, and a laboratory breadboard model of the instrumentation will be constructed. Redox Potential The observation that redox potential will change with metabolic activity is well established. It is common knowledge to the microbiologist that the development of bacteria in broth cultures brings about reducing conditions. In the field of dairy microbiology, various dye reduction tests (methylene blue, resazurin) have been used as indicators of microbial activity in milk. The Marquardt Corporation has been engaged in the development of a biochemical fuel cell for another NASA group, and as a result of these activities made the suggestion that redox potential could be used to follow the metabolic activities of microorganisms in a planetary environment. Their present biodetector cells employ a calomel reference half-cell as the non-biological cathode and a platinum electrode as the biological anode. Sensitivity of the technique appears to be low and the time for a detect- able change in potential to occur is rather long (e.g., 107 to 108 organisms and 4 hours, respectively). Laboratory studies are in progress. Isotopic Tracers Another possible method of life detection is that of phosphorus incorpora- tion into metabolizing cells. The original suggestion, by Harold Morowitz of Yale University, was to use a radioactive isotope of phosphorus as a tracer, but the half lives of the radioactive isotopes of phosphorus of interest are too short for the time span of a planetary mission. This problem can be obviated by use of phosphate labelled with O18. If this phosphate is incorporated, HaO18 would be formed and the process could be followed by monitoring the O16 to O18 ratio in water vapor samples.

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Life Detecting Instruments 501 In a related investigation, the Research Institute for Advanced Studies (RIAS) at Baltimore, has ascertained the laboratory feasibility of using isotopic oxygen anion exchange reactions as a means of life detection. Any living system based on water would, almost certainly, possess the ability to catalyze the exchange of oxygen between water and those oxygen anions that are important in metabolism. The availability of 90 atom per cent excess O1S and of water depleted in O1S would permit a high sensitivity in the detection of these exchange reactions. Such an assay for living systems can be accomplished, in principle, without knowing anything of the composition of the catalysts or the metabolism that the catalysts make possible. The assay is nothing more than an examination of the ability of aqueous extracts or homogenates of extraterrestrial surface material to catalyze the exchange of oxygen between water, phosphate, sulphate, silicate, RCOOH, and possibly, other oxygen anions at rates greater than those catalyzed by specified background conditions. A closely related approach is based on the possibility that nitrogen plays a significant role in Martian life. If nitrogen is an important element in the build-up of living matter, or if nitrogen compounds are involved in energy transformations, one would expect gaseous nitrogen to be released and fixed in some parts of the biological cycle. The stable isotope N16 may be useful for detecting such reactions. Both of the approaches outlined above would require the use of mass spectrometric techniques that permit sampling of the aqueous solution during an incubation period. Gas Exchange Although the techniques now under study and development for the detection of life on other planets make the initial assumption that any existing biota may metabolize specific materials that are well known on Earth, several groups have suggested that it may be possible to detect extraterrestrial life as it exists on other planets without the introduction of foreign or extraneous substances. Terrestrial life requires the conversion of fuel to energy, and this is most likely to occur through an oxidation, or equivalent, process. Hence, in this process gases are usually exchanged. The chemistry of living organisms, while extremely complex, invariably shows a gas exchange. Such gas exchange would permit an inference that metabolizing organisms are present. This, coupled with the output of energy, would allow a determination of the presence of life. It is assumed that metabolism generates energy and liberates heat, that metabolism is a necessary indication of life, and that metabolism is common to all life. Thus, one could detect these activities by mixing the extraterrestrial soil with water to form a culture medium. This would be divided into two

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502 APPENDIX I portions: one portion would be sterilized and used as a control, the second portion would be followed with devices to measure the production of heat and the ratio of N/CO2 in the gas phase. Automated Biological Laboratory It has been recognized by many scientists associated with exobiology that it will eventually be necessary to place large, highly integrated labora- tories of instruments on the surfaces of the planets in order to answer with assurance the question of the presence of life. The problems of design and function of such automated laboratories are under study.