Effect of extraterrestrial environment on bacteri

Hagen, C. A.

English

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R e p o r t  No ,  I I T R I - L 6 0 2 3 - 3  
( Q u a r t e r l y  S ta tus  Report) 
LIFE I N  EXTRATERRESTRIAL ENVIRONMENTS 
Contract No .  NASr-22  
N a t i o n a l -  A e r o n a u t i c s  and 
Space Administration 
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I I T  R E S E A R C H  I N S T I T U T E  
https://ntrs.nasa.gov/search.jsp?R=19660005782 2020-03-16T21:51:54+00:00Z
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Report No. IITRI-L6023-3 
(Quarterly Status Report) 
LIFE IN EXTRATERRESTRIAL ENVIRONMENTS 
August 15 to November 15, 1965 
National Aeronautics and Space Administration 
Contract No, NASr-22 
IITRI Project L6023 
I. INTRODUCTION 
Bacillus cereus spores exposed to either 100 or 13OOC 
for as long as 30 min were not remarkably sensitive to a 
modified Martian environment with diurnal freezing and thawing 
cycles. Although vegetative cell growth and sporulation were 
delayed, population densities after 28 days of exposure compared 
favorably with previous results from nonheated spores. 
Exposure of B. cereus and subtilis spores to a simulated 
Martian environment with barometric pressures over the 5 to 12 mm 
Hg range indicated that the growth response was unusually 
delayed or inhibited when compared to the growth response of 
these organisms in a similar environment but with a pressure 
of 75 mm Hg. 
Manometric studies were initiated to determine the extent 
of adaptation and/or damage that an organism's respiratory 
system undergoes as a result of prolonged contact with a 
simulated extraterrestrial environment., Determination of 
suitable systems were investigated and included: phosphate 
buffer and glucose concentrations, the effect of 1-alanine and 
I I T  R E S E A R C H  I N S i l T U T E  
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manganese sulfate as germinating agents, and different densities 
of B. cereus and B, subtilis spore suspensions, 
11. EXPERIMENTAL PROCEDURES 
The Bacillus species used were the IITRI strain of B. 
subtilis and a B. cereus strain isolated from California desert 
soil. The spore suspensions, prepared in the manner previously 
described, were heat-shocked for  10 min at 8OoC immediately 
before use. 
Experiments investigating the effect of heat upon the 
growth response of B. cereus and the effects of barometric 
pressure upon the growth response of B. cereus and B. subtilis 
were conducted, The environmental conditions for both studies 
were: a daily freeze cycle of 8 hr, a felsite/limonite soil 
containing 1% organic medium, and 7 to 9% moisture. 
For the heat studies the tubes containing the soil were 
inoculated with 0.01 ml of spore suspension and were placed in 
a hot air oven preset at the desired temperature. When the 
tubes equilibrated to the preset temperature (in 5 to 6 rnin), 
they remained at this temperature for 15 or 30 min and were 
then removed and rapidly cooled in an ice water bath. The 
75-mm Hg pressure was established after the addition of water 
to give the required 8 to 9% moisture. 
I l l  R E S E A R C H  I N S T I T U T E  
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8 The pressure studies utilized 5-, 9-? 12-, and 75-mm Hg 
barometric pressures, 
25OC under barometric pressures of 5, 9, and 12 mm Hg, the 
moisture in these tubes was frozen in a dry ice-acetone bath 
prior to the establishment of pressure, 
conducted to maintain the moisture at a constant level although 
it was realized that when the frozen moisture in the tubes 
thawed, the water vapor contributed to the pressure of the 
system in accordance with Dalton's law of partial pressures. 
Since water is in the vapor state at 
This freezing was 
Total and spore counts were performed in both studies 
immediately and at 1? 2, 7, and 28 days unless otherwise stated. 
In all experiments the recovery medium was trypticase soy 
agar (BBL) .  
- B. subtilis was incubated for 48 hr at 3S0Co 
B, cereus was incubated for 24 hr at 35OC, and 
Manometric studies with B, cereus and B, subtilis spore 
suspensions were conducted according to the techniques set 
forth by Umbreit, Burris, and Stauffer,' The harvesting and 
washing of the spore suspensions were described in previous 
reports. 
111. RESULTS A.ND DISCUSSION 
The growth responses of B. cereus spores after exposure 
to dry heat at 100 and 13OOC for 15 and 3 0  min are shown in 
Figures 1 and 2, In all experiments, less than 50% of the 
spores survived the initial heat treatment. There was a 
Urnreit, Burris, and Stauffer? "Manometric Techniques" 4th Ed. 7 J- 
Burgess Publishing Co, ? Minneapolis, 1964, 
I I T  R E S E A R C H  I N S T I T U T E  
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Figure 1 
EFFECT O F  DIURKAL FREEZE-THm CYCLES ON BACILLLS CEREUXJ SPORES 
INITIALLY TREATED W I T I !  DRY FIEAT AT 1 0 0 ° C  M R  15 AND 30 MINUTES 
Experimental  Condi t i c n s :  15 m. oxygen, 8% mois ture ,  
d i u r n a l  temperature  c y c l e  
(-65 to 25'C) with 8 hr 
f r e e z e  cycle. 
l o 6  
lo5 
c I uni.eated, t o t a l  c unt  
o---o Lr.i.edted, spore count  
15 mir.utes h e r t  t rea tment ,  t o t d l  counr- 
&--A 15 mir,utes heat t rea tment ,  spore count  
11---0 30 minutes t e a t  t rea t - ren t ,  t o t a l  c0ut.t 
0--4 30 minutes  heat Lrea 'vent ,  spore cOLin+ 
l o 3  L- I I- I 
0 1 2  7 28 
Days 
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Figure  2 
I,.- 
EFFECT O F  DIURNAL FREEZE-TifAW CYCLES ON BACILLUS CEREUS SPORES 
INITIALLY TREATED WITH DRY HEAT AT 1 3 O o C  FOR 15 AND 30 MIXUTES 
. 
Experimental C o n d i t i o n s :  1 5  mm oxygen, 9k m o i s t u r e ,  
d i u r n a l  temperature cycle 
(-65 t o  25OC) w i t h  8 iu: 
freeze cycle. 
cour: t 
couri?. 
cocln: 
Colin? 
Dd y s  
5 
delayed growth response noticed f o r  both vegetat ive c e l l  growth 
and sporulat ion of spores heated a t  100 and 13OoC, compared t o  
t h e  unheated cont ro l  cul ture .  
t h e  unheated spores occurred between t h e  second and t h i r d  day 
i n  the  simulated environment (not shown i n  Figures 1 and 21, 
while the  vegetat ive c e l l  growth of t h e  spores heated a t  100 
and 13OOC d id  not  reach maximum vegetat ive c e l l  growth a f te r  
28  days of exposure t o  the  environment. 
Maximum vegetat ive c e l l  growth of 
Sporulat ion w a s  s imi l a r ly  delayed i n  the  heated spores, 
The unheated spores reached maximum sporulat ion between 3 and 
7 days,  and the spores heated a t  both 100 and 1 3 O O C  appeared t o  
reach maximum sporulat ion af ter  28  days of exposure. 
Extension of these heat  s t u d i e s  t o  include 16- and 20-hr 
f reeze  cycles would be of i n t e r e s t  s ince  extending the freeze 
cycle  length delays o r  depresses t h e  growth response of the 
bacter ia l  organisms. 
The r e s u l t s  of reduced barometric pressures  upon the 
growth response of B. cereus a re  presented i n  Figure 3 .  W i t h  
o ther  condi t ions being s i m i l a r ,  a lowering of the barometric 
pressure t o  9 o r  5 mm Hg resu l ted  i n  an unusually delayed 
growth response of B, cereus,  This growth inh ib i t i on  m a y  be 
s i g n i f i c a n t  because B. cereus spores germinate with subsequent 
vegetat ive growth i n  the absence of oxygen. The s i g n i f i c a n t  
f a c t o r  appears t o  be the a v a i l a b i l i t y  of w a t e r  s i n c e  water i s  
i n  t he  vapor s t a t e  above 2 and 14OC a t  the reduced barometric 
pressures  of 5 and 9 mm Hg. If w a t e r  i n  t he  vapor phase w e r e  
I I T  R E S E A R C H  I N S T I T U T E  
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Figure 3 
EFFECT OF BAROMETRIC PRESSURE ON THE GROWTH RESFCESE 
OF BACILLUS CEREL'S IN A SIMULATED MARTIAN Eh'VIROI<MEYT 
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Experimental Conditions: No flus:,ing of tu'Les prior to sealing, 
7% moisture, diurnal temperature cycle 
(-65 to 25OC) with 8 hr freeze cycle. 
:- ::m ~ ! g  i.,kro:-LeLric pressure, total ::ou!1- 
c !tun HG t<:rornetric presscre, spore coar,t 
9 r n 9  ny barometric pressure, total count 
*: mm Ha barometric pressure, spore count 
75 K~III i:q barometric pressure, total count 
75 nun HS tarometric pressure, spore count 
O----O 
M 
&--a 
I1---c3 
0 - - I 3  
0 1 2  7 28 
Days 
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I b not available to the organism. the growth cycle of that organism 
would be confined to the region below the temperature maxima of 
2 or 14*C, depending upon the barometric pressure. 
Figure 4 presents similar results of the growth response 
of B, subtilis in a simulated Martian environment with barometric 
pressure reduced to 12 mm Hg, The growth response of this 
organismwasnegative with a slight loss in viability over the 
28-day exposure period, The results with B, subtilis are not 
as significant as those with B. cereus since it has been 
demonstrated previously that 5 subtilis spores require at least 
10-mm Hg partial pressure of oxygen for spore germination and 
vegetative cell growth. 
If the above results from reduced barometric pressures are 
confirmed, they will focus t n e  probieiti UI iLt= pi-e-te~ti~ri ef 
contamination on Mars upon two large groups of microorganisms, 
wild or mutant types: (a) psychrophilic organisms and (b) 
halophiles, or, more generally, those organisms having a low 
water activity ( A  1 requirement for growth, W 
Another ramification of these results would be that if 
growth were delayed rather than inhibited, the massive or 
explosive type of bacterial contamination on a planet like Mars 
could not occur as otherwise was indicated by studies performed 
at higher barometric pressures, 
I I T  R E S E A R C H  I N S T I T U T E  
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Figure 4 
EFFECT OF EAROMETRIC PRESSURE Oh' THE GROWTli RESPOSSE 
OF BACILLUS SJBTILIS  Inl A SIIKLATED MARTIA' EXVIR0:;MEXT 
Experimental Conditions:  No f l u s h i n g  of 1-u'bes p r i o r  t o  s e a l i n g ,  
9% moisture ,  d i u r n a l  temperature cyc le  
(-65 to 25OC) w i t k  8 h r  f r e e z e  cq-cle. 
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f 7 5  mn Hg Larometric p r e s s u r e ,  t o t a l  count 
7 5  mm H g  tarometric p r e s s u r e ,  spore count 
12 mm Eg barometr ic  p r e s s u r e ,  total counc 
1 2  11311 Hg b a r o n e t r i c  p r e s s u r e ,  spore count 
f o---o 
&--a 
- - - - 
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0 1 2  
Days 
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L Superimposed upon the above problem areas is the complete 
lack of knowledge regarding the Martian soil's moisture content 
and moisture retention at different depths, factors which have 
significant bearing on studies of this nature, 
2 
By utilizing the techniques of Levinson and Hyatt, 
spore germination, emergence, and cell division of spores produced 
in severe environments can be studied, 
also be used to study nonsporulating bacteria in order to 
determine whether a cellls respiratory system is adapted or is 
damaged as a result of prolonged contact with a severe environment. 
Similar techniques can 
Manometric studies with B, cereus investigated the influence 
of 0.05 and 0.01 M phosphate buffers at pH 7,O with 5 and 
0.5 mg/ml of glucose as substrate, Heat shocked cereus 
spore suspensions of 3 x lo6  and 3 x lo8 spores/ml did not 
respond favorably to these systems even with the addition of 
0-01 M 1-alanine and trace amounts of manganese sulfate, 
Respiration rate hliters o /lo6 cells/min) were not greater 2 
than the respiration rates of the endogenous control flasks. 
Respiration rates of B. subtilis spores at concentrations of 
3 x 10 and 3 x 10 spores/ml were measurable over a 3 hr- 
period when a complex medium was used as the substrate. 
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pp. 773-788, 1963. 
Levinson and Hyatt, Ann. N ,  Y. Acad. Sci,, V o l .  102, Art. 3 ,  
I l l  R E S E A R C H  I N S T I T U T E  
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IV, s u m y  
B, - cereus spores exposed to 100 and 1 3 O O C  dry heat exhibited 
a delayed response in vegetative cell growth and sporulation 
in a simulated Martian environment compared to nonheat treated 
spores . 
A reduction of the barometric pressure to 5 or 12 mm Hg 
delayed, or inhibited, spore germination and subsequent vegetative 
cell growth of B. cereus. Reduction of the barometric pressure 
to 9 mm Hg produced similar results with B, subtilis spores. 
We believe that it was the lowering of the barometric pressure 
that caused the decrease of available water, especially in the 
case of B. cereus, 
Manometric studies initiated with B, cereus and B. subtilis 
spores investigated suitable substrates for future experiments, 
which will study adaptation or damage to a cell's respiratory 
system as a result of prolonged contact with a severe environment. 
V. PERSONNEL AND RECORDS 
Experiments were planned with the counsel of Dr. E, 
J. Hawrylewicz and with the technical assistance of Miss 
Marjorie Wing and Miss Vivian Tolkacz. 
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Experimental data are recorded in Logbooks 
C16394. 
Re spec tf u l l y  
IIT RESEARCH 
C16249 and 
submitted, 
INSTITUTE 
Approved by: 
Charles A. Hagen 
Associate Bacteriologist 
Life Sciences Research 
Life Sciences Research 
CAT3 :ms 
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C o p y  No.  
D i s t r i b u t i o n  L i s t :  
C o p y  N o ,  Rec ip ien t  
1 - 25 O f f i c e  of G r a n t s  and R e s e a r c h  C o n t r a c t s  
O f f i c e  of Space Sciences 
N a t i o n a l  A e r o n a u t i c s  and Space A d m i n i s t r a t i o n  
Washington 25, D O C ,  
A t t e n t i o n :  SC 
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27 
28 
29 
30 
31 
32 
33 
D r ,  I rv in  D a v i s  
USAF school of A e r o s p a c e  Med ic ine  
P. 0, BOX 4213 
B r o o k s  Air Force B a s e ,  Texas 78235 
D r ,  C ,  S, P i t t e n d r i g h  
D e p a r t m e n t  of Biology 
P r i n c e t o n  U n i v e r s i t y  
P , O ,  B o x  704 
Pr ince ton ,  New Jersey 
D r ,  A l l a n  B r o w n  
D e p a r t m e n t  of Botany 
U n i v e r s i t y  of M i n n e s o t a  
Minneapolis,  M i n n e s o t a  
D r ,  E ,  C, P o l l a r d  
V i s i t i n g  Professor of B i o p h y s i c  
Pennsylvania  State U n i v e r s i t y  
U n i v e r s i t y  Park, Pennsylvania 
D r ,  N o r m a n  H o r o w i t z  
B i o l o g y  D e p a r t m e n t  
C a l i f o r n i a  I n s t i t u t e  of Technology 
Pasadena, C a l i f o r n i a  
D r ,  Melvin C a l v i n  
Space Sciences Laboratory 
U n i v e r s i t y  of Cal i forn ia  
B e r k e l e y  4, C a l i f o r n i a  
D r .  Sidney Fox 
Florida State U n i v e r s i t y  
Tallahassee Florida 
D r ,  C a r l  Sagan 
H a r v a r d  College Observatory 
C a m b r i d g e  38, M a s s a c h u s e t t s  
IIT R E S E A R C H  I N S T I T U T E  
13 
C o p y  N o ,  R e c i p i e n t  
34 D r ,  D a l e  S m i t h  
Ames R e s e a r c h  C e n t e r  
Nat ional  A e r o n a u t i c s  and Space A d m i n i s t r a t i o n  
Moffett Field, C a l i f o r n i a  
O f f i c e  of space Sciences 
N a t i o n a l  A e r o n a u t i c s  and Space A d m i n i s t r a t i o n  
Washington 25, D,C, 
Jet Propulsion Laboratories 
C a l i f o r n i a  I n s t i t u t e  of Technology 
Pasadena, C a l i f o r n i a  
35 D r  D a l e  Jenkins 
36  D r ,  R, E ,  C a m e r o n  
37 D r ,  C a r l  B r u c h  
O f f i c e  of Space Sciences 
Nat ional  A e r o n a u t i c s  and Space A d m i n i s t r a t i o n  
Washington 25, D O C ,  
38 I I T  R e s e a r c h  I n s t i t u t e  
D i v i s i o n  L F i l e s  
39 I I T  R e s e a r c h  I n s t i t u t e  
Editors,  Main F i l e s  
I I T  R E S E A R C H  I N S T I T U T E  
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Life in extraterrestrial environments Quarterly status report, 15 Aug. - 15 Nov. 1965

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