APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1978, p. 730-7370099-2240/78/0035-0730$02.00/0Copyright i 1978 American Society for Microbiology

Vol. 35, No. 4

Printed in U.S.A.

Response of Terrestrial Microorganisms to a SimulatedMartian Environment

TERRY L. FOSTER,* L. WINANS, JR., R. C. CASEY, AND L. E. KIRSCHNER

Science Research Center, Hardin-Simmons University, Abilene, Texas 79601

Received for publication 8 January 1976

Soil samples from Cape Canaveral were subjected to a simulated Martianenvironment and assayed periodically over 45 days to determine the effect ofvarious environmental parameters on bacterial populations. The simulated envi-ronment was based on the most recent available data, prior to the Vikingspacecraft, describing Martian conditions and consisted of a pressure of 7 milli-bars, an atmosphere of 99.9% CO2 and 0.1% 02, a freeze-thaw cycle of -65°C for16 h and 24°C for 8 h, and variable moisture and nutrients. Reduced pressure hada significant effect, reducing growth under these conditions. Slight variations ingaseous composition of the simulated atmosphere had negligible effect on growth.The freeze-thaw cycle did not inhibit growth, but did result in a slower rate ofdecline after growth had occurred. Dry samples exhibited no change during the45-day experiment, indicating that the simulated Martian environment was nottoxic to bacterial populations. Psychrotrophic organisms responded more favor-ably to this environment than mesophiles, although both types exhibited increasesof approximately 3 logs in 7 to 14 days when moisture and nutrients were

available.

As man began to explore the planets, it wasrecognized that there is a need to prevent con-tamination of planets under investigation (4).One type of experiment that has proven usefulin meeting planetary quarantine constraints hasbeen the study of various microbes in simulatedplanetary environments. As early as 1953, Strug-hold (15) performed such investigations as re-lated to Mars, and since that time numerousinvestigators have performed such studies, withthe simulated environments being updated asnew information about Mars became available(2, 3, 6-10, 12, 14, 16-18). Most of these investi-gations demonstrated that microbes can survivethe simulated Martian environment (3, 7, 18),and some demonstrated that a few microorga-nisms can grow if moisture is present (2, 8-10).Because of these and other findings, the Vikingspacecraft were subjected to dry-heat decontam-ination prior to launching in the summer of 1975to reduce the probability of contamination ofMars.Evidence concerning the environment ofMars

has progressed rapidly during the last few years.At one time, all evidence was based on Earth-based experiments (13), but as the space pro-gram developed, more accurate information wasgained by sending spacecraft into the vicinity ofMars (5, 11). There appears to be a void ofresearch dealing with simulated Martian envi-ronments since data from Mariner 9 resulted in

revised concepts of the Martian environment,including higher CO2 concentration, little or noN2, possibly available liquid water, and lowerpressure (5). Also, none of the previous investi-gations used microbial samples from actualspacecraft environments. Because microbesfrom spacecraft areas have the best opportunityto contaminate spacecraft to Mars, it seemedappropriate to include such samples in this in-vestigation. The objective of this investigationwas to determine the response of microorga-nisms isolated from spacecraft areas to a simu-lated Martian environment, based on the mostrecent data from Mars. To expand findings ofthis type of investigation to say that terrestrialorganisms can or cannot grow on Mars is impos-sible, but such studies can provide scientific datato assist in determining the probability of con-tamination of Mars. The Viking spacecraft,which landed on Mars on 20 July and 3 Septem-ber 1976, have supplied data to demonstrate thatresults from Mariner 9 were quite accurate (Sci-ence, vol. 194, 1976). The early results and en-vironmental parameters used by previous inves-tigators are shown in Table 1. Investigators priorto this used primarily a N2 atmosphere at am-bient pressure in a broth medium (14, 16, 17).The environmental parameters used in the pres-ent investigation most closely approximate theactual measurements obtained from the surfaceof Mars.

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GROWTH IN A SIMULATED MARTIAN ENVIRONMENT 731

TABLE 1. Chronological comparison ofparameters used in investigations of simulated Martianenvironments and current data of the Martian environment

Freeze-thaw cy-

Reference Year Gaseous at- (mm of ce Substrate MoistureHg) Temp Time

(OC) (h)9 1967 100.0% air or 10-98 25 8 49.5% felsite 5% RH"

37-100% CO2 -65 16 49.5% limonite1.0% AC medium

18 1967 Air 760 25 4.5 Native soil suspension-75 19.5 in broth

8 1968 37-67% C02 10-98 25 16 49.5% felsite 49% RH30-21% Ar -65 8 49.5% limonite 94%27-13% N2 1.0% AC medium 95%

or 25 8100.0% CO2 -65 16

10 1972 80.0% CO2 5.3 28 8 40:40:10:5:5, felsite-li- 3.8% RH20.0% Ar -60 16 monite-garden soil-

peat-volcanic rock

Current investigation 99.9% CO2 5.3 24 8 Native terrestrial soil Air dried to 10%based on Mariner 9 0.1% 02 -65 16 or 40:40:10:5:5, fel- (wt/wt)

or site-limonite-garden99.0% C02 soil-peat-volcanic1.0% O2 rock

Viking 1 data at land- 95.0% CO2 5.8 -84 Tentatively: Al 2-7%, <0.1%, and proba-ing site 1976-77b 2-3% N2 -28 Si 15-30%, Ca 3-8%, bly abundance of

1-2% Ar Ti 0.25-1.5%, Fe hydrated min-0.1% 02 12-16%, others erals

others-trace

a'RH, Relative humidity.6 Science 194:57-105 (1976); National Geographic 151:3-31 (1977); Scientific

reports released periodically.

MATERIALS AND METHODS under the desigroups were p

Selection of soil samples. Soil samples were ob- various paran

tained from numerous different sites at Cape Canav- ment, such as

eral from areas associated with the Viking spacecraft. ture, nutrient.These samples were obtained by scraping away the sisted of driedfirst 0.5 inch (ca. 12.7 mm) of soil, then collecting the of water addenext 3 to 4 inches (ca. 76 to 102 mm) with a sterile sisted of stuwscoop. Samples were placed into airtight, sterile, plas- (native organtic containers, returned to our lab for study, and stored experimentalat refrigerator temperature prior to use. Fresh samples soil mixture i

were obtained periodically, and all samples consisted soil mixture Mprimarily of sand (many of these sites are built up tained 3% yewith fill sand dredged from the ocean). The only major Martian soildifference between the samples was the amount of soil isolates worganic humus. Many were white sands, containing selected for u

little organic humus, while others were dark sand. In facultativelysome investigations, individual soil samples were sub- set of experimjected to the artificial Martian environment, but in (light and daithe majority of investigations soil samples were thor- 20 min beforoughly mixed, and this composite was subjected to environment.various parameters of the artificial Martian environ- response of niment. tian environnc

Simulated Martian environment. Native Cape for assays ofCanaveral soil samples (1.0 g or 0.1 g) were placed into sealed understerile 10-ml Virtis screw-cap lyophilizer vials (Virtis vented adaptACo., Gardiner, N.Y.). These were subjected to the side an envisimulated Martian environment (Table 2) and sealed Kansas City,

American 237:52-61 (1977); and other Viking

;ired atmosphere. Different experimentalprepared to investigate the effects of theneters of the simulated Martian environ-pressure, incubation temperature, mois-

ts, and gas mixture. Moisture levels con-

i soil or dried soil with 0.01, 0.1, or 1.0 mlad per g of soil. The nutrient levels con-

dies with dark sand versus light sandkic humus versus little organic humus),Martian soil mixture (Table 2), Martianwith 1% peptone (wt/wt), and Martianvith 1% Trypticase soy broth which con-

aast extract. In investigations with themixture, 24-h cultures of Cape Canaveralvere inoculated into the vial. The isolatese was a gram-positive, psychrotrophic,anaerobic sporeforming rod. In anothernents, native Cape Canaveral soil samplesrk sand) were heat shocked at 80°C forre being subjected to the experimentalThis was done to determine the growthative sporeformers in the simulated Mar-nent. Numerous replicates were preparedf each experimental group. Vials were

the desired conditions by use of Virtis-ters attached to a multiport manifold in-ironmental chamber (Labconco Corp.,,Mo.). Screw caps were applied to the

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stoppered vials, and the caps were secured with plasticsealing bands. All vials maintained vacuum conditionsfor the 45-day duration of the experiments. Aftersealing, the vials were immediately placed into thedesired incubator to begin the alternate freeze-thawcycle shown in Table 2. After being prepared, 10 wereremoved immediately to assay the original population.Control vials were prepared in identical fashion butwere ultimately sealed to consist of ambient pressureand/or ambient atmosphere, some being incubated at24°C, others in the freeze-thaw cycle. Appropriatesoils used in these experiments were stored in thesimulated Martian atmosphere at room temperaturefor 24 h before use. The atmosphere was continuouslycirculated through drying towers to assure that driedsamples remained dry. Almost all phases of this inves-tigation were performed at least twice, with manybeing performed three to four times to verify repeat-ability.

Population assays. Ten samples were assayedimmediately for determination of original population,followed by assays of triplicate vials on days 1, 2, and4 and approximately every 3 days for the duration ofthe experiment. Samples were assayed 4 h after re-moval from the -65°C incubator. Each count con-sisted of an average of six plates (triplicate vials platedin duplicate). Samples were diluted in 1% peptone,with the original dilution blank being subjected tosonic disruption for 2 to 4 min (Bransonic model 32Ultrasonic Bath) to disrupt clumps. Subsequent dilu-tions were throughly mixed on a Vortex mixer. Sam-ples were plated by the pour-plate method using Tryp-

TABLE 2. Composition of the simulated Martianenvironment

Component Amt

Atmospherea Variable: 80% C02, 20% Ar or 99%C02, 1% 02 or 99.9% C02, 0.1%02

Pressure 7 millibars (5.3 mm of Hg)

Temperature 24°C day, -65oCb night

Time 8-h day, 16-h night

Duration

Soil

Moisture

Nutrients

45 days

Native Cape Canaveral soil or soilmixture of 40:40:10:5:5, felsite-limonite-garden soil-peat-vol-canic rock

Variable, ranging from dried soilto excess moisture (1.0 ml ofwater per g of soil)

Variable, from no added nutrientsto dehydrated Trypticase soybroth

ticase soy agar (BBL). Preliminary investigations ofplating methods showed no significant difference atthe 0.05 probability level between the spread-plateand pour-plate procedures (T. L. Foster and L. Win-ans, Jr., 1974, Semiannual Progress Report no. 4,NASA CR 139390). Plates were incubated at 7°C for14 days to determine the response of psychrotolerantpopulations and/or at 32°C for 48 to 72 h to determinethe response of the mesophilic bacterial population.All procedures in preparing and assaying samples wereperformed in laminar flow cabinets.

RESULTSPressure. The effect of reduced pressure on

growth of organisms in mixed Cape Canaveralsoils is shown in Fig. 1. These samples containedmoisture and added nutrients to insure thatpressure was the limiting growth factor. The24°C incubation was to serve as a control forviability of organisms in the sample; the graphshows a slower and smaller population increasein the samples under reduced pressure until day14, after which there was no significant differ-ence. The samples incubated in the Martianfreeze-thaw cycle exhibited increases in popula-tion, with the samples under reduced pressureagain showing a slower and smaller increase. Apoint-by-point t test comparison of the freeze-thaw experimental samples shows a significantdifference (0.05 probability level) beginning onday 4, with the reduced pressure resulting insignificantly lower populations. Because of theseresults, all future experiments included a pres-sure of 7 millibars as one of the parameters ofthe simulated Martian environment.Atmosphere. A comparison of ambient at-

mosphere and simulated Martian atmosphere of99.9% CO2 plus 0.1% 02 is shown in Fig. 2. Thesesamples contained excess nutrients and moistureto insure that the atmosphere was the limitinggrowth factor. Populations under both atmos-pheres grew very well, showing similar rates ofincrease for the first 11 days. After this time, thesamples in the ambient atmosphere showed bet-ter growth until day 42, at which time the pop-ulations in the simulated atmosphere appearedto recover and again start increasing. Both ex-perimental groups showed approximately a 3-logincrease in population during the 45 days of theexperiment. Control groups incubated at a con-stant 24°C (Fig. 2) showed a faster initial in-crease, a slightly higher population (approxi-mately 0.5 log), and a faster rate of decline.A comparison of ambient and simulated Mar-

tian atmospheres using dry soils was also per-formed. The results showed static populationsfor the, 45-day duration of the experiment, anda t test comparison of the final populations andthe initial populations showed no significant dif-ference (0.05 probability level).

a Obtained as analyzed mixtures in single cylindersfrom Carroll Chemical and Cryogenics, Abilene, Tex.

' Revco ULT 1175, West Columbia, S.C.

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CORSTANT 24'C

0 --o REDUCED PRESSURE

* .-- 0 ANRIERT PRESSURIE

FREE ZE-TNAW

0-.o REDUCER PRESSURE

* . SANRERTC PRESSURE

SI 14 ;IR 11 14 UoRAYS IRCURATIOR

FIG. 1. Effect of reducedpressure (7 millibars) on growth of soil microorganisms incubated at 240C and in

a fr-eeze-thaw cycle (-650C for 16 h; 240C for 8 h) in a simulated Martian environment consisting of99.9% CO2

plus 0.1% 02 with supplemental moisture and nutrients.

PIRIEZE-THAW CONSTANT 24 C

0~~~~~~~~

~~~~~~~~~00 3..0 4-.0

b

rinamshr09.%C2pu

and~

Because of questions concerning the proposedMartian atmnosphere, numerous earlier experi-ments were performed using two other experi-mental atmospheres, one consisting of 80% 002

plus 20% argon, the other consisting of 99% 002

plUS 1% 02. The population changes observed in

those earlier experiments followed identical

trends to those shown in this investigation.These results demonstrate that the growth re-

sponse is virtually the same in the three gaseous

atmospheres used in these investigations.Incubation temperature. The effect of in-

cubation temperatures of the samples is also

shown in Fig. 2. The population changes shown

on the left are a result of incubation of the

samples under the simulated Martian freeze-

thaw cycle of 8 h at 2400 followed by 16 h at

-650C. The curve on the right shows identical

conditions except with incubation at constant

2400. As should be expected, the initial popula-tion increases occurred more rapidly in the sam-

ples incubated at 2400; however, the populationsincreased by approximnately the same amount.

The significant observations are that the sam-

ples under the ambient atmosphere maintained

their high populations longer in the freeze-thaw

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incubation, and the samples under the simulatedMartian atmosphere appeared to recover in thefinal days in the freeze-thaw incubation. Thisindicates that incubation of samples at constant24°C results in a faster rate of decline oncemaximum populations have been achieved. Theresults shown in Fig. 1 also tend to verify thepopulation changes shown in Fig. 2, i.e., popu-lation increase is more rapid at 24°C, approxi-mately the same population is achieved, and therate of decline is more rapid at 24°C.Moisture and nutrients. Detailed investi-

gations concerning the effect of moisture onresponse of bacterial populations to the simu-lated Martian environment have not been com-pleted sufficiently to estimate the minimumamount of water required for growth. However,all experiments performed thus far included aset of soil samples that had been dried andstored under a dry atmosphere and a set con-taining added moisture (0.1 ml/g of soil). Anexample of such a comparison is shown in Fig.3. It can be seen that the dry samples showed nochange in populations, whereas the samples withexcess moisture showed a 1-log increase whichwas maintained for the duration of the experi-ment. The population increase with added mois-ture in Fig. 3 was not as great as that shown inFig. 2, but the samples in Fig. 2 also containedadded nutrients. This comparison, then, showsthat available nutrients will have a definite effecton growth oforganisms in the simulated Martianenvironment.

In other experiments, pure cultures of CapeCanaveral soil isolates were inoculated into ar-tificial Martian soil (Table 2) with varying

0

> 7

2

St

00S.

amounts of moisture and nutrients. Growth innative Cape Canaveral light sand (little organichumus) was also compared with that in darksand. Because of the large number of growthcurves generated in this broad study, the resultsare tabulated and presented in Table 3. Eachnumber is an average of six plate counts, and allexperiments were performed at least twice toverify results. As can be seen, population in-creases were greater in the dark sand, whichcontained more nutrients. In the presence of theartificial Martian soil, population increases wereinsignificant until nutrients were added, and in-creases were greater with a richer supply ofadded nutrients.None of the experiments demonstrated

growth when the soil samples were dried. On theother hand, none showed a decline in the popu-lations of the dry samples, indicating that thesimulated Martian atmosphere is not toxic toorganisms in the samples. There was littlegrowth when 0.01 ml of water per g was added,more of an increase with 0.1 ml of water. Ofthese combined results, the greatest populationincreases occurred with the richest nutritionalsupply and greatest amount of water. It alsoappeared that less water was required when anative soil sample was used than when the arti-ficial Martian soil was used. For this reason,most experiments with native soil where mois-ture was not to be a limiting factor had 0.1 ml ofwater added per g of soil.

Similar results were demonstrated for a gram-positive, non-sporeforming isolate from CapeCanaveral soil (data not shown). The majordifference was that this isolate demonstrated a

-~~~ . . . . . . . . . . ..

0 S 10 15 20 25 30 35 40 45

DAYS INCUBATION

FIG. 3. Effect of soil moisture on growth ofmicroorganisms incubated in a freeze-thaw cycle (-65°C for 16h; 24°C for 8 h) in a simulated Martian environment (99.9% CO2 plus 0.1% 02 at 7 millibars pressure). Excessmoisture indicates 0.1 ml of water added per g of soil.

0 - - ---

* 0

i o°--- 0-0 o

v ~~~~-0 00 0a

0 0

o-o DRY SOIL

*---------EXCESS MOISTURE

9 ,

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GROWTH IN A SIMULATED MARTIAN ENVIRONMENT 735

TABLE 3. Response of bacterial populations in experimental soils grown in a simulatedMartian environmenta

Moisture Days to

Soil (ml of wa- Additional nutri- Original counthb Maximum Total log maxi-ter per g of ents (wt/wt) count increase mum

soil) count

Cape Canaveral, light sand Dried 3.1 x 104 3.1 x 104 21Cape Canaveral, light sand 0.1 3.1 x 104 2.3 x 105 0.87 21Cape Canaveral, dark sand Dried 5.8 x 104 5.9 X 104 0.01 21Cape Canaveral, dark sand 0.1 5.8 x 104 3.0 x 107 2.7 14Artificial Martian soil Dried 3.0 x 104 3.0 x 1i0 21Artificial Martian soil 0.01 3.0 x 104 5.5 X 104 0.26 7Artificial Martian soil 0.1 3.0 x 104 9.0 x 1i0 0.48 4Artificial Martian soil 1.0 3.0 x l04 3.3 X 104 0.04 14Artificial Martian soil Dried 1% peptone 3.0 x 104 3.1 x 104 14Artificial Martian soil 0.01 1% peptone 3.0 x 10W 9.0 x 104 0.48 21Artificial Martian soil 0.1 1% peptone 3.0 x 104 2.0 x 105 0.82 4Artificial Martian soil 1.0 1% peptone 3.0 X 104 1.4 x 106 1.67 14Artificial Martian soil Dried 1% TSBc 3.0 x 104 2.9 x 104 21Artificial Martian soil 0.01 1% TSB 3.0 X 104 9.3 x 105 1.49 4Artificial Martian soil 0.1 1% TSB 3.0 x 104 7.3 X 106 1.39 2Artificial Martian soil 1.0 1% TSB 3.0 x 104 1.7 x 108 3.75 21Heat-shocked light sand Dried 6.6 x 103 6.6 x 103 21Heat-shocked light sand 0.1 6.6 x 103 3.1 x 106 2.68 7Heat-shocked dark sand Dried 2.7 x 104 2.8 x 104 21Heat-shocked dark sand 0.1 2.7 x 104 1.4 x i0W 3.71 7

a Native Cape Canaveral soil, with dark sand and with little (light sand) native organic humus, and artificialMartian soil (40:40:10:5:5, felsite-limonite-garden soil-peat-volcanic rock), grown with various moisture andnutrient levels in a simulated Martian environment (99.9% CO2 plus 0.1% 02 at 7 millibars at -65°C for 16 h and24°C for 8 h).

b Colony-forming units per gram of sample; each count is an average of six plates.c Trypticase soy broth supplemented with 3% yeast extract.

decrease in population in the dry samples, butrequired less nutrients to grow well (greater than3 log increases) when moisture was present atmaximum concentration.Table 3 also shows the results obtained when

the light and dark Cape Canaveral soil sampleswere heat shocked before use to eliminate thenon-sporeforming populations. By comparingthe increases in populations of these samplesand of the non-heat-shocked samples, it can beseen that the sporeforming population appearsto respond more favorably to the simulated en-vironment than does the total population. Again,the populations did not change over 21 days inthe dried samples, but showed -significant in-creases within 7 days with added moisture.Assay incubation temperature. In assay-

ing for population changes in these experiments,plates were incubated at both 7 and 24°C. Thechanges in populations showed the same trendsunder both incubation conditions, except thatthe psychrotolerant populations increasedslightly faster, by comparison, than the meso-philic populations. Controls used throughoutthese investigations included samples performedas described for the experimental sets, but in-cubated under ambient pressure, temperature,

and/or atmosphere. These were performed asviability controls, and in no case did populationdecreases occur.

DISCUSSIONIt is recognized that simulated environmental

studies cannot duplicate all physical, chemical,and atmospheric parameters that constitute agiven planetary environment. Even though theparameters used in this investigation were basedupon the most current available data, approxi-mate the environmental conditions of Marsmore closely than earlier investigations, and aresimilar to actual results obtained from the Vi-king program, it is understood that there arelimits to such a study. Nevertheless, such aninvestigation can provide results based upon sci-entific evidence about certain factors which mayinfluence or limit growth in a given environment.As indicated earlier, these experiments were per-formed a minimum of two times, most of themat least four to five times, with the results beingrepeated in each instance. It is felt that theresults indicate definite trends of bacterial pop-ulation response to various parameters of thesimulated Martian environment.

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Although the conditions of this simulatedMartian environment were different from thoseof earlier investigators, some of the results pre-sented here are consistent with results presentedby several earlier investigators. For example,Young et al. (16-18) demonstrated that orga-nisms grow well in the Martian freeze-thaw cy-cle, and this was shown in our results.Although the Viking spacecraft show a colder

temperature than those used in this investiga-tion, the landing sites for the Viking landerswere selected on the basis of factors other thantemperature. The freeze-thaw cycle of this studyrepresents an optimum temperature of Marswith reference to terrestrial organisms. Becauselow temperature and the freeze-thaw cycle werenot lethal to the bacterial populations, it is fea-sible to assume that terrestrial contaminantscould be introduced onto the Martian surface ina less than favorable temperature environment,survive, and later be blown by strong Martianwinds to a more optimal temperature environ-ment. To make further extrapolations of thistype, at least two other questions would have tobe answered. First, how long could terrestrialcontaminants (especially sporeformers) survivethe extreme cold of a nonoptimum site? Sec-ondly, what is the minimum high temperatureof the freeze-thaw cycle required for growth tooccur, and for what period of time must thistemperature be maintained to actually result inincreases in the bacterial population? Althoughthese questions are not answered by the presentinvestigation, it is apparent that a continuedextremely low temperature will retard growth,and that a favorable temperature must bereached before growth can occur.This investigation also agrees with the work

of those who found growth in a simulated Mar-tian environment (2, 8-10, 16), those who statethat moisture and nutrients are the most likelylimiting factors of growth (2, 8-10), and thosewho state that a dry Martian environment willresult in survival of terrestrial microorganisms(2, 6, 7, 12, 14). The diversity of simulated Mar-tian environments used in these types of exper-iment places emphasis on the tremendous adap-tibility of microorganisms.Probably the most often mentioned factor re-

sponsible for restricting growth of terrestrial or-ganisms in the Martian evnironment is the avail-ability of liquid water. However, the results ofthe Viking program cite the presence of morewater on Mars than predicted (Viking 1, Earlyresults, 1976, NASA SP-408, National Aeronau-tics and Space Administration), and a model isproposed where liquid water may be available atpressures below 6 millibars when the tempera-ture reaches 00C. The results of this investiga-

tion demonstrate that growth can occur in waterlevels as low as 1% (wt/wt) if nutrients areavailable. Although this is a much higher waterlevel than measured on Mars, it is emphasizedthat the proposed liquid water model wouldmake water available in microenvironments, andthe water concentration could reach a level ap-proximating this.The factor that is possibly the most important

for restricting growth of terrestrial organisms inthe Martian environment is available nutrients.Although results are not complete, the Vikinglanders have not yet detected organic com-pounds in the Martian soil. Because the mostlikely contaminants on such a spacecraft wouldbe heterotrophic sporeforming organisms, theywould require organic nutrient sources. Also,elements required in trace amounts for growthof other types of microoganisms may not befound; if so, this would preclude growth of ter-restrial organisms.

Several significant conclusions can be drawnfrom this investigation. The reduced pressure ofthe Martian environment does have an effect ongrowth of microorganisms. The populationchanges were approximately the same in allthree of the experimental Martian atmospheres,and growth occurred in all three when moisturewas added. In dry samples, the populations wereunchanged for the duration of the experiments,demonstrating that the simulated Martian en-vironment was not toxic to the organisms. Thisindicates that the lack of moisture should notprevent prolonged survival of terrestrial orga-nisms. The freeze-thaw cycle of this investiga-tion allowed growth of microorganisms and usu-ally resulted in a slower rate of decline thanincubation at 24°C. Organisms capable ofgrowthat low temperatures appeared to respondslightly more favorably to the simulated Martianenvironment. This is of significance because ofthe recent data demonstrating the presence ofpsychrotrophic organisms in Cape Canaveralsoils (1). Populations in heat-shocked soil sam-ples appeared to respond more favorably in theexperimental Martian environment than non-heat-shocked populations. The sporeformerused in this investigation was more resistant tothe dry environment and required less water togrow than the non-sporeformer. These last tworesults are of significance because of dry-heatsterilization of the lander spacecraft. Because ofthe results from the Viking landers indicatingno organic compounds in the soil, and the pres-ent results demonstrating the requirement oforganic compounds as a nutrient source, lack ofnutrients is possibly the limiting growth factoron Mars.By terrestrial standards the environment of

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GROWTH IN A SIMULATED MARTIAN ENVIRONMENT 737

Mars is hostile, and data from the Viking landerswill likely support this concept. However, theresults of this and numerous other investigationsemphasize the adaptability of microorganisms.Therefore, based upon current information, noenvironmental model ofMars has been proposedthat would preclude growth of terrestrial orga-nisms on the surface of Mars, although the prob-ability of such growth is admittedly small.

ACKNOWLEDGMENTSThis investigation was supported by the National Aeronau-

tics and Space Administration under grant NGR-44-095-001.

LITERATURE CITED

1. Foster, T. L., and L. Winans, Jr. 1975. Psychrophilicmicroorganisms from areas associated with the Vikingspacecraft. Appl. Microbiol. 30:546-550.

2. Fulton, J. D. 1960. Survival of terrestrial microorganismsunder simulated Martian conditions, p. 606-613. In 0.0. Benson, Jr. and H. Strughold (ed.), Physics andmedicine of the atmosphere and space. John Wiley &Sons, Inc., New York.

3. Hagen, C. A., E. J. Hawrylewicz, and R. Ehrlich.1967. Survival of microorganisms in a simulated Mar-tian environment. Appl. Microbiol. 15:285-291.

4. Hall, L. B., and R. G. Lyle. 1971. Foundations of plane-tary quarantine. Environ. Biol. Med. 1:5-8.

5. Hartmann, W. K., and 0. Raper. 1974. The new Mars,the discoveries of Mariner 9. NASA SP-337. NationalAeronautics and Space Administration, Washington,D.C.

6. Hawrylewicz, E., B. Gowdy, and R. Ehrlich. 1962.Microorganisms under a simulated Martian environ-ment. Nature (London) 193:497.

7. Hawrylewicz, E., C. Hagen, and R. Ehrlich. 1965.Response of microorganisms to a simulated Martianenvironment, p. 64-73. In M. Florkin (ed.), Life sciencesand space research, vol. 3. North-Holland PublishingCo., Amsterdam.

8. Hawrylewicz, E., C. Hagen, V. Tolkacz, B. Ander-

son, and M. Ewing. 1968. Probability of growth ofviable microorganisms in Martian environments, p.146-156. In A. H. Brown and F. C. Favorite (ed.), Lifesciences and space research. North-Holland PublishingCo., Amsterdam.

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