AUTOMATED

LIFE-DETECTION

FOR THE VIKING

MISSION

EXPERIMENTS

TO MARS

H A R O L D P. K L E I N Ames Research Center, NASA, Moffett Field. Calif. 94035, U.S.A.

Abstract. As part of the Viking mission to Mars in 1975, an automated set of instruments is being built to test for the presence of metabolizing organisms on that planet. Three separate modules are combined in this instrument so that samples of the Martian surface can be subjected to a broad array of experimental conditions so as to measure biological activity. The first, the Pyrolytic Release Module, will expose surface samples to a mixture of C140 and C14Oz in the presence of Martian atmosphere and a light source that simulates the Martian visible spectrum. The assay system is designed to determine the extent of assimilation of CO or CO2 into organic compounds. A small amount of water can be injected into the gas phase during incubation upon command. The Gas Exchange Module will incubate surface samples in a humidified CO2 atmosphere. At specified times, portions of the incubation atmosphere will be analyzed by gas chromatography to detect the release or uptake of CO2 and several additional gases. A rich and diversified source of organic nutrients and trace compounds will be available as further additions to the incubating samples. The Label Release Module will incubate surface samples with a dilute aqueous solution of simple radioactive organic substrates in Martian atmosphere, and the gas phase will be monitored continuously for the release of labeled CO2. Each module, in addition to its gas and nutrient sources, incubation chambers, and detector systems, contains heaters capable of sterilizing surface samples to serve as controls. Since the instrument is designed to operate under Martian conditions and to detect Martian, not terrestrial, organisms, and because the final flight instruments can perform only four assays for each module, formidable problems exist in testing the hardware. The implications of this situation are discussed.

1. Introduction The p l a n e t M a r s has been the object of speculation concerning the existence o f extraterrestrial life for a long time. D u r i n g the p a s t h u n d r e d years, scientific interest has w a x e d a n d w a n e d as new i n f o r m a t i o n accrued a b o u t the planet. A f t e r the flight o f M a r i n e r 4 in 1964, a n d M a r i n e r s 6 a n d 7 in 1969, estimates o f the p r o b a b i l i t y o f life on M a r s were pessimistic ( H o r o w i t z , 1971) b a s e d in p a r t on the d a t a r e t u r n e d b y these spacecraft. However, M a r i n e r 9, which has been orbiting M a r s since N o v e m b e r 1971, revealed t h a t p l a n e t to be m u c h m o r e c o m p l e x t h a n has b e e n a s s u m e d a n d also cons i d e r a b l y m o r e interesting as a t a r g e t in the search for extraterrestrial life ( M a r i n e r M a r s , 1971). I n the late s u m m e r o f 1975, two m o r e U.S. spacecraft are p l a n n e d for l a u n c h to Mars. These spacecraft, c a r r y i n g identical p a y l o a d s , constitute the Viking mission. T h e y will also o r b i t the p l a n e t for p r o l o n g e d p e r i o d s but, o f m o r e interest in connection with u n d e r s t a n d i n g h o w far chemical e v o l u t i o n has p r o c e e d e d on M a r s , they will deliver two landers to the surface, a p p r o x i m a t e l y 6 weeks apart. Tentative p r i m a r y l a n d i n g sites have a l r e a d y been selected - the first one being at a b o u t 2 0 ~ and 34~ within a large basinlike area, the second, at 4 4 ~ a n d 10~ within a latitude b a n d where the highest quantities o f water are regularly observed in the M a r Origins of Life 5 (.1974) 431-441. All Rights Reserved Copyright 9 1974 by D. Reidel Publishing Company, Dordrecht-Holland

432

HAROLD P. KLEIN

tian atmosphere and where liquid ground water may exist for brief periods (Farmer, 1973).

2. General Considerations about Viking Biology Experiments Many of the scientific experiments on the Viking lander should contribute, either directly or indirectly, to answering questions about the current stage of chemical evolution of Mars (Soften and Young, 1972). Of these, only the so-called 'direct biology' investigation is described here, the instrumentation for which is based on the concepts and experimental work of a group of biologists * who have been formulating this aspect of the mission for several years. This group early established a number of general principles that have guided the development of the designs for the Viking Biology Instrument (VBI). First, it was agreed that, with the current lack of information about the Martian surface, together with almost total ignorance about local environmental 'niches', and because of the extreme importance of the question being addressed, a m i x of experiments was imperative, based on different assumptions about the characteristics of Martian organisms. Of the many techniques for 'life detection' under development at that time (Bruch, 1966), those based on metabolic measurements were considered the most desirable for an initial mission. Second, the initial search was to be for microbia! soil organisms since, based on terrestrial analogy, these should be most ubiquitous, most adaptive, and most hardy as a group. Third, samples were to be obtained from the upper few centimeters of the soil** where both chemosynthetic and photosynthetic forms might be available. Sampling, furthermore, was to be conducted several times during the lifetime of the landers to take maximal advantage of any seasonal variations in accessibility to Martian organisms. Samples were to be incubated at temperatures no higher, and preferably lower, than the highest known temperatures anywhere on Mars (approximately 35 ~ The incubations were to extend over periods of several days. Finally, it was stipulated that the capability to sterilize soil samples - to serve as controls in the event of positive results - must be incorporated into the experiment. The deliberations of this group interacting with the mission planners resulted in the final choice of three separate biological experiments (discussed below). Each experiment is based on different assumptions about Martian biota. In addition, they incorporate a number of internal options, thus further extending the flexibility of the combination.

3. VBI Experiments Samples for the VBI will be acquired several times during each 90-day Viking lander mission through the use of a surface sampler that will also serve other experiments, * The Viking Biology Team consists of N. Horowitz (Cal. Tech.), H. P. Klein (Ames), J. Lederberg (Stanford Univ.), G. Levin (Biospherics Corp.), V. I. Oyama (Ames), A. Rich (M.I.T.), and W. Vishniac (Univ. of Rochester). ** The term 'soil' throughout this discussion denotes the Martian surface material. The precise nature of the surface material is, of course, unknown at this time.

AUTOMATED LIFE-DETECTION EXPERIMENTS FOR THE VIKING MISSION TO MARS

433

including an inorganic analysis experiment (Toulmin et al., 1972) designed to yield data on the elemental composition of the soil, and an organic analysis experiment (Anderson et al., 1972). Sulface samples will enter the VBI through a sieving device so that particles larger than 2 mm will be excluded. The soil will pass into a soil SOIL DISTRIBUTION SCHEMATIC LABELED RELEASE

GAS EXCHANGE

] PYROLYTIC

I

COMMONSERVICES

Fig. 1. Schematicdrawing of the three Viking biology modules. distribution assembly within the VBI, which will then automatically deliver measured volumes of soil to each of the three experiments. The latter are to be housed in individual modules, which together with a common services module and an electronics subassembly, comprise the VBI. Figure 1 is a schematic drawing of these modules (see also Figure 7).

4. Pyrolytic Release (PR) Experiment The PR experiment (Horowitz et al., 1972) is designed to measure either photosynthetic or chemosynthetic fixation of CO z or CO. The main rationale for this is that the Martian atmosphere consists primarily of COz, with CO as a trace component, and that the Martian biota would include organisms capable of assimilating one or both of these gases. Furthermore, it seems reasonable that a sustained biota on Mars would include photosynthetic organisms. The PR experiment incubates soil in a Martian atmosphere to which ~*CO 2 and a*CO are added and then, by pyrolysis and the use of an organic vapor trap, determines whether a4C has been fixed into organics. The experiment can be conducted in the dark or in light.

434

HAROLD P. KLEIN

~ H20VAPOR ~ HELIUM ~MARS GASES ~ 14CO2/14CO ~ 1 SOIL

OFF B DETECTOR

~PYROLYSIS

DUMP CELL E~ H20

VEN"

HOLDING CHAMBER

VENT

Fig. 2. Schematic drawing of the Viking Pyrolytic Release experiment. At this stage of the sequence, the soil sample is ready to be incubated in the presence of radioactive gasses. Within the PR experiment are three test cells, at least one of which may be used for more than one soil sample. In the test cell reserved for use as a control, the soil is first heated at 160~ for 3 h and then incubated. For an analysis, 0.25 cc of soil is loaded into a test cell, which is then moved to the incubation station and sealed. Figure 2 is a diagram of this experiment at this stage. After establishing the incubation temperature of 15~176 one of the PR options may now be exercised. Water vapor can be introduced by ground command or omitted. Then 20/tl of a mixture of J4COz (957oo)-14CO(57oo) are provided from a gas reservoir, and a xenon arc lamp (12 V, 6-W maximum power) would normally be turned on. However, the option exists to command this lamp not to be turned on during the ensuing incubation, which lasts for 5 days. After incubation, the test cell is heated to 120 ~ to remove the residual incubation gases that are vented to the outside. Background counts are made at about this time in the sequence, after which the test cell is moved from the incubation station to the pyrolysis station. As shown in Figure 3, pyrolysis is accomplished by heating the test cell to 625 ~ while purging the test cell with helium. The purged gases pass through the organic vapor trap (OVT), packed with Chromosorb P coated with CuO, into a 14C detector. Since the OVT is designed to retain organic compounds and fragments, the 1~C detector at this stage will sense a 'first peak' consisting mainly of unreacted (bound?) 14COz/~4CO (and also some 14COz from decarboxylation reactions that occur during pyrolysis). This first peak is regarded as nonbiological in origin. After this operation, the test cell is moved away from the pyrolysis station, the detector is heated and purged with helium, and background

435

AUTOMATED LIFE-DETECTION EXPERIMENTS FOR THE VIKING MISSION TO MARS

]H20 ~

VAPOR HELIUM

]MARS

HELIUM SUPPLY

GASES

W

14CO2/14C0

l

SOIL

OFF

D T TDR

WINDOW \ INCUBATION HEAD END " ~ .

-14CO2/14CO RESERVOIR 400 MB

PYROLYSIS 120~

20/xl

DUMP CELL

i

H20

VENT

HOLDING CHAMBER

VENT

Fig. 3. Pyrolytic Release experiment. At this stage of the analysis, the incubation chamber has been moved to the pyrolysis station. counts are taken once more to verify that the background radiation is down to prepyrolysis levels. The organics are then released from the OVT by heating it to 700 ~ at which time they are simultaneously oxidized to CO2. As these are flushed into the t4C detector, a second radioactive peak at this point would indicate biological activity. A series of cleanup operations follows these steps, bringing the PR module to a stage of readiness for the next sample. 5. Gas Exchange (GE) Experiment This experiment (Oyama, 1972) measures the production or uptake of CO 2, N > CH 4, H2, and 02 during the course of incubation o f a Martian soil sample by means of gas chromatography. The GE experiment can be conducted in one of two modes: in the presence of water vapor, without added nutrients, or in the presence of a complex source of nutrients. The first option is based on the assumption that substrates may not be limiting in the Martian soil (Hubbard et al., 1971) and that biological activity may be stimulated when water becomes available. The second option assumes the presence of significant numbers of anaerobic heterotrophs in the Martian soil. For the GE experiment, only a single test cell is available, but this can be used sequentially for a number of samples if necessary. The test cell can also be heated to 160~ for 3 h to serve as a control. After receiving 1 cc of soil from the distribution assembly, the test cell is moved to the incubation station and sealed. After a helium purge, a mixture of helium, krypton,

436

HAROLD P. KLEIN GAS EXCHANGE EXPERIMENT ~

He/Kr/C02

~

REFERENCE COLUMN

HELIUM

~ ~

lO0,u.I SAMPLETUBE

MARS GASES LIQUIDNUTRIENT

COLUMN m

VENTS2~----~

TESTCELL GASES

-

-

SOIL

He SUPPLY

HELIUM

Fig. 4. Schematic drawing of the Viking Gas Exchange experiment incubated in the 'humid' mode. and CO 2 is introduced and this becomes the initial incubation atmosphere*. At this point, the option exists to introduce either 0.5 or 2.5 cc of the nutrient solution provided for this experiment. Using the lesser quantity, as in Figure 4, the soil does not come into contact with the solution and incubation proceeds in a 'humid' mode. An additional 2.0 cc allows contact between the soil and the nutrients. The latter consists of an aqueous mixture of d- and 1-amino acids, vitamins, other organic compounds, and inorganic salts. As currently planned, incubation at 15 ~ + I0 ~ (in the dark) will initially be in the humid mode for 7 days, after which additional nutrient solution is added by command. For gas analyses, 100/d of the atmosphere above the soil are removed through the use of a gas sampling tube. This occurs at the beginning of each incubation and after 1, 2, 3, 5, 9, and 15 days. The sampled gas is placed in a stream of helium flowing through the chromatograph column (25 ft long, packed with 100-120 mesh Poropak Q) into a thermal conductivity detector. The system used in the GE experiment separates the gases of interest with a resolution of at least 95~ between adjacent peaks and detects changes of the order of 1 nanomole over the range of 1 to 1000 nanomoles, except for hydrogen which is detectable in the range of from 10 to 10000 nanomoles. After a 15-day incubation cycle, the option exists to add a fresh soil sample to the test cell and begin a new incubation cycle or to drain the medium from the test cell, replacing it with fresh nutrients, and also replacing the original atmosphere with * Krypton serves here as an internal standard, and helium is used to bring the pressure in the test chamber to approximately 200 rob.

437

AUTOMATED LIFE-DETECTION EXPERIMENTS FOR THE VIKING MISSION TO MARS

flesh incubation atmosphere. The latter procedure will be used if gas changes are noted in the initial incubation, on the assumption that if these changes are due to biological activity, they shouJd be repeatable and also be enhanced, while if of nonbiological origin, they should not reappear.

6. Label Release (LR) Experiment The LR experiment (Levin, 1972) is designed to test metabolic activity in a soil sample moistened with a dilute aqueous solution of 14C.labeled simple organic compounds. The rationale for this experiment is that Martian organisms should be in equilibrium with the atmospheric CO2 on Mars and that at least some of them should be able to L A B E L E D RELEASE EXPERIMENT

DETECTOR

HELIUM SU~PPL~

VENT

HEAD END

LABELED NUTRIENT RESERVOIR

15 •176

~

H20 VAPOR

~

HELIUM

~TEST

H20 RESERVOIR

CELL GASES

~

MARSGASES

I

SOIL

LIQUID NUTRIENT HELIUM SUPPLY

Fig. 5. Schematic drawingof the Viking Label Releaseexperiment. catabolize organic compounds to CO 2. The experiment then depends on the biological release of radioactive gases from a mixture of simple compounds supplied during incubation. For this experiment (Figure 5), one of the four test cells provided receives 0.5 cc of soil sample and is moved to the incubation station and sealed, the Martian atmosphere being established in the test cell in this process. Before the labeled nutrients (a mixture of 14C-formate, 14C-glycine,14C- d- and l-lactate, t4C- d- and 1-alanine, and 14C-glycolic acid; all carbons labeled) are added, a background count is taken, and the nutrient solution is degassed by passing helium through the nutrient reservoir. Approximately 0.15 cc of nutrient is then added, and incubation proceeds in the dark at 15 ~ +__10 ~

438

HAROLD P. KLEIN

for 11 days. The atmosphere above the soil sample is monitored by a 14C detector continuously throughout the incubation, after which the test cell and detector are purged with helium. Additional cleanup operations are performed to bring the remaining radioactivity of the detector down to background levels in preparation for the next analysis. As with the other experiments, the test cells can be heated at 160 ~ for 3 h when a control analysis is to be performed.

7. Testing the VBI Figures 6 and 7 show the VBI brought to a ftightlike configuration. Unlike the instrument shown, however, the actual flight instruments, which will be very similar in appearance, will not be subjected to testing with biological (soil) samples since the final VBI's must be pristine with respect to soil samples. The main reason for this is that each of the final instruments can only test a maximum of four samples, after which some of the consumable supplies or test cell space becomes limiting. Even without these constraints, there would be inherent hazards in subjecting the final flight hardware to terminal heat sterilization procedures after testing with soils

Fig. 6. Viking Biology Instrument (Development unit). Larger box, approximately 1500 cu3, contains the mechanical subsystem including the four modules described in the text. Smaller box conrains the electronic subsystem, which includes sequencers and data processing equipment.

AUTOMATED LIFE-DETECTION EXPERIMENTS FOR THE VIKING MISSION TO MARS

439

Fig. 7. VBI Development Unit with side panels removed. Individual modules can be seen; the inverted U-shaped tube (left center) is the OVT for the PR experiment; the gas chromatograph columns are coiled at top, right,

440

HAROLD P. KLEIN

because unsuspected soil particles might later plug nutrient, vent, or other lines in the VBI or interfere with proper sealing of the test chambers at their incubation stations. Thus, it follows that confidence in the final instruments must rely on systematic prior testing of precursor instruments, like the one shown, together with rigorous control over the manufacture and assembly of each component used in the VBI. In this regard, several difficulties immediately become apparent in devising appropriate test procedures. The most folmidable is that the VBI is designed to operate on Mars and not on Earth. Proper operation of the instrument itself- quite apart from the incubation conditions - requires appropriate thermal and atmospheric environments as well as ambient pressures equivalent to those expected on Mars (i.e., about 5 rob). Another major testing problem stems from the fact that positive biological signals cannot be obtained for all three experiments, even with a properly functioning instrument, from valid positive terrestrial soil samples, because the experiments are designed for Martian, not terrestrial, organisms. Thus, performing the PR experiment on terrestrial soils without water, or even in the presence of the small amount of water vapor that can be commanded in this experiment, would be futile as a test procedure since a body of laboratory evidence exists showing that terrestrial organisms require the frank addition of water before significant CO 2 fixation is observed. Similarly, the GE experiment, when conducted in the humid mode, yields virtually no biologically derived gas changes with terrestrial soils - the latter apparently being substrate-limited, rather than water-limited. Only the LR experiment gives reasonable data with terrestrial soil when these are tested under conditions approximating those to be encountered on Mars. From the above, it is evident that no single soil type, nor any known terrestrial organism, can serve as a standard test object for all three VBI experiments. Consequently, the following procedures have been developed as testing 'standards' to gain confidence in the metamorphosis of the VBI from laboratory 'breadboards', working with terrestrial samples in a terrestrial environment, through several precursor instruments, to the final flight articles. First, a set of test standard gases has been selected for each experiment. For the LR and PR experiments, a known quantity of ~4CO2 of known radioactivity is used as the standard against which the operation of OVT's and 14C detectors can be tested. When injected into the test chambers, the 14COz, of course, serves also to check for leaks. Development of the GE experiment is guided by the use of several known standard mixtures of the gases of interest. These serve to calibrate gas chromatograph column performance and detector performance. In addition to the test standard gases, soil standards are also used. For the PR experiment, a standardized sample of 'Chatsworth' soil, which had previously fixed 14CO2 under optimal (i.e., wet) conditions, is used to test the complete system beginning with pyrolysis. This 'preincubated' soil is remarkably uniform in its properties and is stable over long periods of time when stored dry and at low temperature. Table I illustrates the reproducibility of data obtained in the PR experiment using this preparation. For the LR and GE experiments, another soil (Aiken soil),

AUTOMATED LIFE-DETECTION EXPERIMENTS FOR TIdE VIKING MISSION TO MARS

441

TABLE I PR experiment: performance data and the reproducibility of 'standard' pre-incubated chatsworth soil Run

Pyrolysis peak (lst peak P1)

Organic peak (2nd peak P2)

1

5.8 • 104

2 3 4 5 6

4.8 • 5.9 • 6.1 • 5.6 • 5.5 •

1.77 • 1.43 • 1.71 • 1.70 • 1.69 • 1.69 •

104 104 104 104 104

Mean values 5.3 =k0.45 • 104 (8%)

104 104 104 104 104 104

1.66:k0.1 • 104 (7%)

c o n t a i n i n g a b o u t 10 6 b a c t e r i a per gram, is used to challenge the c o m p l e t e systems. It, too, can be m a i n t a i n e d for long p e r i o d s w i t h o u t any significant change in the biological 'signals' obtained. By the use o f these test s t a n d a r d s , it has in fact been possible to relate the results o b t a i n e d with c o n v e n t i o n a l l a b o r a t o r y e q u i p m e n t to those o b t a i n e d with the h a r d w a r e being built for eventual use on Mars. Several versions o f the VBI a l r e a d y have been m a n u f a c t u r e d a n d tested a n d m o r e will be a s s e m b l e d a n d tested during the e v o l u t i o n o f the final flight h a r d w a r e . A f t e r all the p l a n n e d testing has been successfully accomplished, we will have r e a s o n a b l e assurance t h a t the landed i n s t r u m e n t s will p e r f o r m satisfactorily on Mars. References

Anderson, D. M., Biemann, K., Orgel, L. E., Oro, J., Owen, T., Shulman, G. P., Toulmin, P., III, and Urey, H. C.: 1972, Icarus 16, 111. Bruch, C. W.: 1966, in C. S. Pittendrigh, W. Vishniac and J. P. T. Peterman (eds.), Biology and the Exploration of Mars, PuN. no. 1296, National Academy of Sciences, National Research Council, Washington, D.C., p. 487. Farmer, C. B. : 1973, Icarus, in press. Horowitz, N. H.: 1971, Bull. Atomic Sci. 27, 13. Horowitz, N. H., Hubbard, J. S., and Hobby, G. L.: 1972, Icarus 16, 147. Hubbard, J. S., Hardy, J. P., and Horowitz, N. H.: 1971, Proc. Nat. Acad. Sci. U.S. 68, no. 3, 574. Levin, G. V.: 1972, Icarus 16, 153. Mariner Mars: 1971, Project Science Report, National Space Science Data Center, NASA Goddard Spaceflight Center, Greenbelt, MD. Oyama, V. I.: 1972, Icarus 16, 167. Soften, G. A. and Young, A. T.: 1972, Icarus 16, 1. Toulmin, P , Baird, A. K., Clark, B. C., Keil, K., and Rose, H. J.: 1973, Icarus 20, 153-178.