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UNPtiBUSHED PREUfflHAOT DATfc 

CA/A $A d-K .. /(fij^ortjij) iitri /194-125 OT^ ' 

_JL>[Q uarterly Status Report,)-*— 

LIFE IN EXTRATERRESTRIAL ENVIRONMENTS^- t# i 7 rva ~*^' 

National Aeronautics //** " ^ #-*•'*? 

and Space Administration 
Washington, D.C. 



I IT RESEARCH INSTITUTE 




Report No. IITRI-C194-12 
(Quarterly Status Report) 

LIFE IN EXTRATERRESTRIAL ENVIRONMENTS 

November 15, 1963, to February 15, 1964 

National Aeronautics and Space Administration 



CNfrS f\ Contract No> NASr-32) 
IITRI Project CI 9 4 



I. INTRODUCTION 

The survival of Bacillus subtilis var. qlobiqii in a simu- 
lated Martian environment modified by addition of 1% organic 
medium and moisture concentrations of 0=25, 2.0, 4.9, and 21c 7% 
was studied. The organism survived this environment, but only 
by virtue of its ability to form spores. B. subtilis spores 
placed in the simulated Martian environment modified by (a) 
addition of 10% organic medium plus 8% moisture or (b) addition 
of 10% organic medium plus 16% moisture plus 5% oxygen did not 
germinate during the first freeze-thaw cycle, even when they 
were given prior heat- shock treatment. The decrease observed 
in total and spore counts after 1 and 2 weeks of exposure could 
indicate that the spores germinate but do not survive. 

A strain of Pseudomonas aeruginosa did not survive in the 
simulated Martian environment modified by addition of 10% or- 
ganic medium plus 10% moisture. The death of this organism 
was very rapid during the first thaw cycle. In subsequent 



I IT RESEARCH INSTITUTE 



The fungal component from the lichen Teloschistes 
chrysophthalmus was isolated by washing the lichen thalli in 
cold tap water for 30 min and soaking them overnight in sterile 
distilled water. Fragments were excised from thalli and macer- 
ated in a 25-ml Potter-El vehj em tissue homogenizer containing 
10 ml of sterile water and 0.1 ml of Tween 80. The macerated 
tissue was streaked on the surface of unacidified potato dex- 
trose agar (Difco) and incubated at 20° C for 12 days., Then 1- 
cm squares of the agar containing the mycobiont were aseptically 
transferred to mycological agar (Difco) acidified to pH 4„8 and 
maintained at room temperature. 

The diffusible pigments of lichen and mycobiont were ex- 
tracted from agar with hot 95% ethanol according to the method 

2 

of Asahina and Shibata. 

III. RESULTS AND DISCUSSION 

The survival of B. subtilis in the simulated Martian en- 
vironment with 1% organic medium plus 2% moisture is shown in 
Figure 1. Initially, 70 to 84% of the cells present were spores, 
and after one freeze- thaw cycle essentially all the remaining 
cells were spores. The concentrations of organic medium and 
moisture were not sufficient to support the growth of this or- 
ganism. The number of viable cells recovered from the tubes 

2 

Asahina and Shibata, "Chemistry of Lichen Substances," Tokyo, 

1954. 

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of the experimental group decreased slightly over the 112-day 
exposure period. 

Figure 2 shows the effect of higher moisture levels and a 
1% organic medium on the survival of B. subtilis . Moisture con- 
centrations of 4.9 and 21.7% had an initial effect on survival. 
Greater numbers of bacteria survived the flushing and inoculating 
procedures in the tubes containing the higher moistures „ The 
total count curves show a relationship between the moisture con- 
tent and the high initial death rate. The slight initial in- 
creases in spore counts were perhaps due to the moisture, but 
the decreasing spore counts after 28 days of exposure indicated 
the effect was not a lasting one. 

Figures 3, 4, and 5 show the effect of freezing and thawing 
on Bo subtilis , Ps. aeruginosa , and B. cereus with 10% organic 
medium added to the simulated Martian soil. Plate counts were 
done hourly for the first 3 hr of freezing and first 6 hr of 
thawing. 

Heat-shocked spore suspensions of B. subtilis an d B. cereus 
were used to determine whether these spores are capable of germi- 
nating in the simulated Martian environment, and also whether 
germinated spores multiply and subsequently sporulate. Figure 3 
shows the effect of 0.5% oxygen and increased moisture content 
(16%) on the B. subtilis spore suspension. No effect was noticed 
on the germination potential of the spores through a freeze-thaw 



1 1 T RESEARCH INSTITUTE 



Figure 2 

THE EFFECT OF VARYING CONCENTRATIONS OF MOISTURE 
ON THE SURVIVAL OF BACILLUS SUBTILIS VAR. GLOBIGII 
IN THE SIMULATED MARTIAN ENVIRONMENT 



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0.25% Moisture Total Count 

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£ ^ 4.9% Moisture Total Count 

^. -A 4.9% Moisture Spore Count 

H El 21.7% Moisture Total Count 

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cycle o The decrease in total and spore counts after 1 and 
2 weeks of exposure could indicate that the spores are germi- 
nating but not surviving „ This experiment will be continued 
for 4 months . 

The survival of Ps. aeruginosa in the simulated Martian 
environment with 10% moisture is shown in Figure 4. The or- 
ganisms survived the initial freezing with no significant 
change in the number of viable cells; but a very rapid de- 
crease began 2 hr after thawing was initiated, and 1.4% of 
the cells were recovered at the end of 18 hr» After a 1-week 
exposure both aerobic and anaerobic cell counts had decreased 
to less than 0.02% of the initial count. Qualitative tests 
for oxidase, cytochrome C, and nitrate and nitrite reduction 
did not show any differences between aerobically and anaero- 
bically grown cells. 

The effect of the simulated Martian environment with 20% 
moisture on B. cereus spores is shown in Figure 5. The con- 
centrations of organic medium and moisture had no effect on 
the germination of spores during the initial freeze-thaw 
cycle or after subsequent cycles. Survival of this organism 
will be examined over a 4-month exposure period. 

Several unsuccessful attempts were made to isolate the 

3 4 

lichenized fungus by the spore method of Hale and Ahmadjian. 

3 Hale, Bull. Torrey Bot. Club, 82, 182-187,- 1958. 
4 
Ahmadjian, Bryologist, 64, 168-179, 1961. 

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Successful isolation was achieved "by macerating thalli and 
streaking them on unacidified potato dextrose agar. Hyphal 
fragments germinated in 12 hr. Aspergillus niqer . Penicillium 
spp., and Alternaria spp. were observed as contaminants. Twelve 
days after inoculation the mycobiont produced a yellow, water- 
soluble pigment whose margin in the agar coincided with a dis- 
tinct zone of inhibition to the contaminants. 

Small squares of agar containing the mycobiont were trans- 
ferred to acidified mycological agar. In pure culture the 
colonies of the fungus changed from white to yellowish brown 
within 3 weeks. The fungus produced a large amount of pigment, 
which was observed as droplets on top of the mycelial pad„ 

Since it has not been conclusively demonstrated that lich- 
enized fungi produce reproductive or other taxonomically signi- 
ficant structures, the mycobiont was identified by comparison 
of the pigment it produced with that produced by the parent 
lichen „ 

Absorption spectra of pigments extracted from the parent 
lichen and the mycobiont exhibited an absorption maximum at 
430 mu in the visible range. The absorption in the ultravio- 
let range was very great; the resolution of the curve is being 
determined. The pigment extracted from the parent lichen was 
temperature sensitive and turned clear at room temperature but 
remained yellow at 15°C. 



1 1 T RESEARCH INSTITUTE 



12 



IV. SUMMARY 



fn«s A- 



Incorporation of 4„9 or 21=7% moisture into the dry simu- 
lated Martian soil modified by the addition of 1% organic 
medium increased the number of B subtilis surviving the ino- 
culating and flushing procedures » Lower moisture concentra- 
tions, 2o0 and 0„25%, did not have this effect „ However, the 
death rate was greater in the tubes with 4„9 and 21 „ 7% moisture „ 
Thus, after 56 days of exposure there was no significant differ- 
ence between the groups. 

Bo subtilis spores in Martian environment modified by 10% 
organic medium appeared to be slightly affected by (a) 8% mois- 
ture or by (b) 16% moisture plus 0.5% oxygen,, The decrease in 
total and spore counts indicates that the spores germinate but 
do not survive „ 

Less than 0.02% of Ps „ aeruginosa cells survived a 1-week 
exposure to Martian environment modified by 10% organic medium 
and 10% moisture = 

Bo cereus spores survived the simulated Martian environment 
modified by 10% organic medium plus 20% moisture, but there was 
no apparent germination » 

Isolation of the fungal component from the lichen 
T o chrysophtha lmu s was accompli shed „ A method for identifying 
the mycobiont by absorption spectra of diffusible pigments and 
lichenolic acids has been initiated. ^ jnr ^ ^ f> L 



1 1 T RESEARCH INSTITUTE 
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V. FUTURE PLANS 

A critical examination of the function of moisture on the 
growth of bacteria during constant and diurnal temperatures 
has been initiated in order to establish the minimum environ- 
mental conditions necessary for growth. These studies will be 
expanded to include diurnal temperatures of various durations 
and soils containing different concentrations of moisture plus 
organic medium. 

Methods will be investigated to study the effects of mois- 
ture, organic medium, and temperature on the germination of 
spores in the simulated Martian environment c 

Similar studies will be conducted with T„ chrysophthalmus 
to define the growth- limiting factors inherent to the simulated 
Martian environment „ 

The effect of ultraviolet light on lichen growth and phy- 
siology and on the ability of lichens to imbibe substantial 
quantities of water from ambient atmospheres will be investi- 
gated. 

The program will be greatly stimulated by the acquisition 
of soil and lichen specimens from Antarctica. These specimens 
are being collected by Dr. Rudolph of the Ohio State University's 
Polar Institute. 



I IT RESEARCH INSTITUTE 



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VI 



RECORDS AND PERSONNEL 



The experimental data are recorded in Logbooks C 13795, 
C 14081, and C14419o 

Technical assistance was given by Miss Charlene Berger 

Respectfully submitted, 
I IT RESEARCH INSTITUTE 



Approved by: 




A 



y iMAAjls 



E o Jo/ Ha wry lewi.cz 

Assistant Director 

of Life Sciences Research 



Charles A. Hagen >> 
Associate Bacteriologist 
Life Sciences Research 




Regn^ll Jo/iet^ J r.J4*-*Z\ 
AssistantM^relogist 
Life Sciences Research 



CAH/RJ/cg 



NT RESEARCH INSTITUTE 



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Copy No. 

Distribution List : 

Copy No. Recipient 

1-25 Office of Grants and Research Contracts 
Office of Space Sciences 

National Aeronautics and Space Administration 
Washington 25, D„C 
Attention: SC 

26 IIT Research Institute 
Division L Files 

27 IIT Research Institute 
Editors, Jo Jo Brophy, Main Files 

28 IIT Research Institute 

Ko Wo Miller, Report Library 

29 Dr. Irving Davis 
Major, USAF, MSC 
European Office 

Office of Aerospace Research 
Shell Building, 45 Cantersteen 
Brussels, Belgium 

30 Dr. Co So Pittendrigh 
Department of Biology 
Princeton University 
P 0„ Box 704 
Princeton, New Jersey 

31 Dr. Allan Brown 
Department of Botany 
University of Minnesota 
Minneapolis, Minnesota 

32 Dr. E. Co Polard 

Visiting Professor of Biophysics 
College of Chemistry and Physics 
Pennsylvania State University 
University Park, Pennsylvania 



IIT RESEARCH INSTITUTE 



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Copy No . Recipient 



33 Dr. Norman Horowitz 
Biology Department 

California Institute of Technology 
Pasadena, California 

34 Dr. Kirby- Smith 
Biology Division 

Oak Ridge National Laboratory 

P 0. Box Y 

Oak Ridge, Tennessee 

3 5 Dr. Melvin Calvin 

Space Sciences Laboratory 
University of California 
Berkeley 4, California 

36 Dr, Sidney Fox 
Florida State University 
Tallahassee, Florida 

37 Dr. Carl Sagan 
Department of Astronomy 
University of California 
Berkeley, California 

38 Dr. Dale Smith 
Ames Research Center 

National Aeronautics and Space Administration 
Moffett Field, California 

39 Dr. Dale Jenkins 
Office of Space Sciences 

National Aeronautics and Space Administration 
Washington 25, D.C. 

40 Dr. R. E. Cameron 

Jet Propulsion Laboratories 
California Institute of Technology 
Pasadena, California 



I I T RESEARCH INSTITUTE 



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