Simulated Martian atmospheric and ecological effects on Bacillus cereus spore germinatio

Hagen, C. A.

English

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I -  - 
I -  
Report N o .  I I T R I - L 6 0 2 3 - 5  
( Q u a r t e r l y  S t a t u s  R e p o r t )  
L I F E  IN EXTRATERRESTRIAL ENVIRONMENTS 
Contract No. NASr-22 
National Aeronautics and 
Space Administration 
I l 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=19660016973 2020-03-16T19:21:06+00:00Z
I 
Report No. IITRI-L6023-5 
(Quarterly Status Report) 
I 
LIFE IN EXTRATERRESTRIAL ENVIRONMENTS 
February 15 to May 15, 1966 
National Aeronautics and Space Administration 
Contract No. NASr-22 
I I T R I  Project L6023 
I. INTRODUCTION 
Martian environment simulation experiments are in progress 
to study the effects of the following barometric pressures and 
carbon dioxide concentrations on Bacillus cereus spores: 
Earth atmosphere at pressures of 10, 25, 40, 
and 98 m b ,  with diurnal temperature cycle 
Carbon dioxide concentrations of 37, 67, 
and 100% at pressure of 98 mb, with diurnal 
temperature cycle 
Carbon dioxide concentrations and pressures 
of 37% at 40 m b ,  67% at 25 mb and 100% at 
10 m b ,  with diurnal temperature cycle. 
B. cereus spore germination was inhibited by carbon dioxide 
concentrations of 37, 67, and 10096 at all pressures. 
as low as 10 mb with Earth atmosphere were not inhibitory to spore 
germination, but vegetative cell growth was less than that at 
98 mb. 
Pressures 
i l f  R E S E A R C H  I N S T I T U T E  
1 
II 
8 
b 
S 
I 
I 
8 
I 
8 
8 
I 
Soil ecology experiments were initiated to determine the 
minimum numbers of a bacterium required to survive and grow 
in different soil types. A California desert soil with water 
added to establish an equilibrium relative humidity of 99% is 
being used. Experiments with B. cereus, Pseudomonas aeruqinosa, 
Putrefactive Anaerobe (PA 3679), and Staphvlococcus aureus are 
in progress. 
A l l  organisms, except P. aeruqinosa, survived inoculation 
procedures at 50 to 100% recovery levels: less than 1007 of the 
pseudomonads were recovered. Preliminary results indicated 
that inocula of P. aeruqinosa and S .  aureus of 200 to 300 cells 
per gram of soil do not survive 7 days at either a constant 
3OoC or a diurnal temperature cycle in the desert soil with 
99% relative humidity and 1013 mb Earth atmosphere. 
I1 EXPERIMENTAL PROCEDURES 
P. aeruqinosa ATCC 10145 and S .  aureus ATCC 12411 were grown 
on the surface of trypticase soy with 2% agar for 24 hr at 35OC. 
Maximum spore production was obtained by growing B. cereus on 
TAM (Difco) for 6 days at 35OC and PA 3679 in a 5.0% trypticase 
and 0.5% peptone broth for 12 days at 35OC. The cells were har- 
vested and washed with 0.025 M phosphate buffer, pH 7.0, before 
final suspension in the buffer. The cell suspensions were stored 
i l T  R E S E A R C H  I N S T I T U T E  
2 
in the refrigerator until used. The B. cereus and PA 3679 spore 
suspensions were heat-shocked at 8OoC for 10 min and at 6OoC for 
15 min, respectively, before use. 
The gas mixtures were obtained from the Matheson Company, 
Inc., Joliet, Illinois. Gas 
the following results: 
Tank No. Gas 
1 Carbon dioxide 
Nitrogen 
Argon 
Oxygen 
2 
3 
Carbon Dioxide 
Nitrogen 
Argon 
Oxygen 
Carbon dioxide 
Nitrogen 
Argon 
Oxygen 
ana lyses were performed with 
Amount R e q i  ired, 
% 
100 
0 
0 
0 
67 
13 
20 
0 
33 
33.5 
33.5 
0 
Analvsis, % 
>99 
0 
0 
< 5 Ppm 
66.2 
12.6 
21.2 
5 Ppm 
37 
26.9 
36.1 
< 5 PPm 
An electric hygrometer-indicator (Hygrodynamics , Inc . , Silver 
Springs, Maryland), model 15-300, was used to determine equili- 
brium relative himidity (ERH) . 
California desert soil' (No. 68-3, Mecca Hills, California, 
0.5 to 6-in. deep) with a high clay content was used. In the 
'Soil collected and provided by Dr. R. E. Cameron, Jet Propulsion 
Laboratory, Pasadena, California. 
I l T  R E S E A R C H  I N S T I T U T E  
3 
Martian environment simulation studies, mixtures containing 
different relative amounts of pulverized felsite and limonite 
were used, 
111. RESULTS AND DISCUSSION 
Martian Environment Simulation Studies 
1. Atmospheric Comtmsition and Pressure 
Current estimates of the Martian surface pressure range from 
A *.. 
5 to 40 mb. 
view are related to pressure -- (1) carbon dioxide abundance 
and (2) water availability. 
Two important factors from a biological point of 
With the established carbon dioxide limits of the Martian 
atmosphere by spectroscopic analysis the relative abundance of 
carbon dioxide would range from 100 to 33% over the 5- to 
40-mb pressure range. 
metabolite for bacteria. Normally, it does not appear to be an 
essential nutrient since most heterotrophic bacteria produce it 
in metabolism or utilize the small amount present in air. Some 
species of bacteria require elevated carbon dioxide concentra- 
tions for growth, but there is little information on carbon di- 
oxide toxicity for spore germination or vegetative cell growth. 
Carbon dioxide is regarded as an essential 
Similarly, water availability is affected by barometric pres- 
sure, 
OOC,  but at 40 mb, water exists as a liquid at temperatures as 
high as 3OoC, 
At 5 mb, water exists as a vapor at temperatures above 
With the liquid phase of water i i n i i t e d  "uezai~se 
I l l  R E S E A R C H  I N S T I T U T E  
4 
of pressure, growth of a microorganism would occur only over 
very narrow temperature ranges of the diurnal freezing and 
thawing cycles. 
Figures 1 through 4 show the effect of Earth atxmsshere 
(air) at reduced barometric pressures on B. cereus spores. 
Each tube in the Martian environment simulation studies con- 
tained 1 g of felsite/limonite soil, 1% organic medium and 
8 to 9% moisture. The tubes were flushed seven times with 
air before pressure was established and the tubes were sealed. 
An 8- and a 20-hr diurnal freeze cycle were used with each 
pressure and atmosphere. 
Spore germination and vegetative cell growth, evidenced 
by marked increase in total count, occurred at 10, 40, and 
98 mb and to a less extent at 25 mb during the 8-hr freeze 
cycle: growth occurred between 3 and 7 days. The 8-hr freeze 
cycle total count at 7 days was omitted from Figure 2 because 
of its uncertainty, but values will be obtained at 28 and 56 
days. 
As shown in Figures 5 through 7, pressures of 10, 25, and 
40 mb with respective carbon dioxide concentrations of 100, 67, 
and 37% inhibited spore germination of B, cereus for at least 28 
days during both the 8- and the 20-hr diurnal freeze cycles. A 
loss of viability did not accompany inhitition of spore germination. 
I I T  R E S E A R C H  I N S T I T U T E  
5 
I 
8 
e 
E 
8 
I 
1 
I 
II 
THE EFFECT OF EARTH ATMOSPHERE AT IOmb 
P R E S S U R E  ON BACILLUS C E R E U S  SPORES. 
o-------O 8 HR. FREEZE CYCLE, TOTAL COUNT 
C.---+ 8 HR. FREEZE CYCLE, SPORE COUNT 
20 HR. FREEZE CYCLE, TOTAL COUNT 
&---d 2 0 H R .  FREEZE CYCLE, GPORE COUNT , 
DAYS 
FIGURE I 
6 
THE EFFECT OF EARTH A T M O S P H E R E  AT 2 5 m b  
P R E S S U R E  ON B A C I L L U S  CEREUS SPORES. 
- 
I- 
F 
0- 8 HR. FREEZE CYCLE, 
e--- + 8 HR.'FREEZE CYCLE, 
A - 4  20HR. FREEZE CYCLE, 
&--- i 20HR. FREEZE CYCLE, 
TOTAL COUNT 
SPORE COUNT 
TOTAL COUNT 
SPORE COUNT 
n 
IO+- 
0 1  3 7 
D A Y S  
FIGURE 2 
7 
14 
. 
J 
0 a 
- 
THE EFFECT OF EARTH ATMOSPHERE AT 4 0 m b  
PRESSURE ON BACILLUS CEREUS SPORES. 
I o8 
IO? 
IO6  
A 
AI 
io5 
IO 
- -  8 HR. FREEZE CYCLE, TOTAL COUNT 
- +--- -0 8 HR. FREEZE CYCLE, SPORE COUNT 
- 20 HR. FREEZE CYCLE. TOTAL COUNT 
- It--- 4 2 0 H R .  FREEZE CYCLE, SPORE COUNT 
O i  3 - 
DAYS 
FIGURE 3 
i4 
THE EFFECT OF EARTH ATMOSPHERE AT 9 8 m b  
PRESSURE ON B A C I L L U S  C E R E U S  SPORES. 
I 0 8 t  - - - 
- 
- 
-0 8 HR. FREEZE CYCLE, SPORE COUNT 
CYCLE, TOTAL COUNT A- 20 HR. FREEZE 
&----a 20HR. FREEZE CYCLE, SPORE COUNT 
D---------il CONSTANT 35OC, TOTAL COUNT 
-I CONSTANT 35" C, SPORE COUNT 
I I I 
3 
7 - 2 8  
DAYS 
FIGURE 4 
9 
THE EFFECTOF 100% CARBON DIOXIDE AT IOmb 
PRESSURE ON BACILLUS CEREUS SPORES. 
m z 
8 HR. FREEZE CYCLE, TOTAL COUNT 
e---- 8 HR. FREEZE CYCLE, SPORE COUNT 
2 0 H R .  FREEZE CYCLE, TOTAL COUNT 
&--- 4 2 0 H R .  FREEZE CYCLE, SPORE COUNT 
cn 
LL 
0 
E 
c 
a 
- 
of 
w t 
DAYS 
FIGURE 5 
IO 
THE EFFECTOF 67% CARBON DIOXIDE AT 25m> 
PRESSURE ON BACILLUS CEREUS SPORES. 1 
o------(> 8 HR. FREEZE CYCLE, TOTAL COUNT 
* 8 HR. FREEZE CYCLE, SPORE COUNT 
e20 HR. FREEZE CYCLE, TOTAL COUNT 
4 20HR. FREEZE CYCLE, SPORE COUNT 
0 - 
E (0 W a m 
z I 
z, 1 1041 I I I 
0 1  3 7 28 
I I 
I 
DAYS 
FIGURE 6 
THE EFFECTOF 37% CARBON DIOXIDE A T  4 0 m b  
PRESSURE ON BACILLUS CEREUS SPORES. 
I o6 I 
I O  
0-0 8 HR. FREEZE CYCLE, TOTAL COUNT 
*--- 8 HR. FREEZE CYCLE, SPORE COUNT 
A- 20 HR. FREEZE CYCLE, TOTAL COUNT 
&--- 4 2 0 H R .  FREEZE CYCLE, 6PORE COUNT 
I- 
0 1  3 7 
DAYS 
FIGURE 7 
12 
28 
;I 
I 
I 
I 
8 
I 
Emission spectroscopy for the following elements did not 
show any significant differences between the soils: aluminum, 
arsenic, boron, calcium, chromium, copper, iron, mercury, 
lithium, magnesium, manganese, sodium, nickel, lead, silicon, 
tin, titanium, vanadium, and zinc, 
Mass spectrometry indicated that of the two so i l s  that in- 
hibited spore germination, one had a high argon content and the 
other a high sulfur dioxide content. 
is of importance because of the heat sterilization and the gas- 
Neither of these substances 
- _ - -  LL- siLiy Liiat the S C ~ ~ S  r e ~ ~ i ~ e .  
I l T  R E S E A R C H  I N S T I T U T E  
13 
THE EFFECTOF 37% CARBON DIOXIDE AT 9 8 m b  
PRESSURE ON BACILLUS CEREUS SPORES. 
LL 
0 
]E 
a 
(3 
\ 
K 
0 > 
> 
OL 
3 
a 
IL 
0 
cn 
W 
- 
a 
m z 
3 
z 
t- - 8 HR. FREEZE CYCLE, TOTAL COUNT 
*--- 8 HR. FREEZE CYCLE, SPORE COUNT 
A- 20 HR. FREEZE CYCLE, TOTAL COUNT 
&---A 2 0 H R .  FREEZE CYCLE, SPORE COUNT 
0 1  3 7 
DAYS 
FIGURE 8 
14 
28 v -  
THE EFFECT OF 67% CARBON DIOXIDE AT 9 8 m b  
PRESSURE ON BACILLUS CEREUS SPORES- 
I o7 
I o6 
A 
A 
I o5 
io4 
0 
- - 8 HR. FREEZE CYCLE, TOTALCOUNT 
- e--- * 8 HR. FREEZE CYCLE, SPORE COUNT 
- A-A 2 0 H R .  FREEZE CYCLE, TOTAL COUNT 
&--- 1. 20  HR. FREEZE CYCLE, SPORE COUNT - 
I I 
I 3 7 
DAYS 
15 
P a r t i c l e  s i z i n g  of t h e  so i l s  showed t h a t  w i th  f u r t h e r  ad- 
d i t i o n  of pu lver ized  l imoni te ,  t he  percentage of  particles less 
than  44 p i n  diameter  increased,  S o i l  t h a t  permitted normal 
spore germination and vege ta t ive  ce l l  growth had 65% of 
particles less than 44 p i n  diameter as compared wi th  a poor 
s o i l  t h a t  had 81% of its particles less than  44 p i n  diameter. 
its 
B. S o i l  Ecoloqy S t u d i e s  
T e s t s  w e r e  started by using tubes conta in ing  1 g of desert 
s o i l ,  99% r e l a t i v e  h m i d i t y ,  and a i r  a t  1013 mb.  The tubes 
w e r e  inocula ted  w i t h  d i l u t e d  c u l t u r e  suspensions of P. aeruqinosa 
and S, aureus t o  g i v e  200, 2 0 ,  2 ,  and 0.2 cel ls  per gram of soil. 
The tubes w e r e  sealed and placed a t  either a cons t an t  3OoC o r  a 
d i u r n a l  temperature c y c l e  (-65 to  25OC), 
w e r e  cracked open, and trypticase soy b r o t h  w a s  added. The tubes  
w e r e  incubated a t  37OC f o r  7 days,  and t h e  con ten t s  w e r e  s t reaked  
on t h e  s u r f a c e  of t r y p t i c a s e  soy agar. P. aeruqinosa and S .  aureus 
could no t  be recovered from any of t h e  tubes. 
A t  7 days t h e  tubes 
Another series of experiments with higher  ce l l  concen t r a t ions  
are i n  progress .  P. aeruqinosa w a s  inocula ted  a t  10 3 , 10 4 , and 
2 
ce l l s  per g r a m  of so i l ,  and B. ce reus  w a s  inocula ted  a t  10 , 5 10 
lo3 ,  and 10 
t h e  same environmental condi t ions  descr ibed  above. Pre l iminary  
r e s u l t s  i nd ica t ed  t h a t  99% of B. c e reus  cel ls  b u t  less than 10% 
of P o  aeruqinosa cel ls  survived inocu la t ion  procedures. 
4 cells per gram of so i l .  All tubes w e r e  placed under 
I l T  R E S E A R C H  I N S T I T U T E  
16 
IV. SUMMARY 
Carbon dioxide concentrations of 37, 67, and 100% at pres- 
sures of 40, 25, and 10 rnb ,  respectively, inhibited B. cereus 
spore germination during 8- and 20-hr diurnal freeze cycles. 
Inhibition of B. cereus- spore germination also occurred in 
atmospheres with 37 and 67% carbon dioxide at 98 mb during both 
freeze cycles. 
B. cereus spore germination was not inhibited in air at re- 
duced pressures of 10, 25, and 40 mb, but the growth response 
was less than that at 98 mb. 
Low numbers of P. aeruainosa and S. aureus did not survive 
7 days in a desert soil environment with 99% relative humidity, 
air at 1013 m b ,  and either a constant 3OoC or a diurnaltemper- 
ature cycle. 
These data will be presented at the forthcoming COSPAR 
meeting (May 12, 1966), Vienna, Austria. Reprints of the 
paper will be distributed when available. 
V. PERSONNEL AND RECORDS 
Experiments were planned with the consel of Dr. E. J. 
Hawrylewicz and the technical assistance of Miss Marjorie 
Ewing, Mrs. L. G. Larson, and Miss Vivian Tolkacz. 
I l T  R E S E A R C H  I N S T I T U T E  
17 
E x p e r i m e n t a l  data  are recorded i n  I I T R I  Logbooks C 1 6 6 7 8 ,  
C16684, C 1 6 6 8 9 ,  C16876, and C16882 ,  
1 
Respectfully s u h i t t e d ,  
I I T  RESEARCH INSTITUTE 
Associate B a c t e r i o l o g i s t  
L i f e  Sciences R e s e a r c h  
Approved by: 
I 
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U 
L i f e  Sciences R e b e a r c h  
I I T  R E S E A R C H  I N S T I T U T E  
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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 :  
COPY No.  
1 - 25  
26 
27 
28 
29 
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R e c i p i e n t  
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 
National 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. 
A t t e n t i o n :  SC 
D r .  I rv in  D a v i s  
USAF School of A e r o s p a c e  Medicine 
P.O. B o x  4213 
B r o o k s  Air Force B a s e ,  Texas 78235 
D r .  C. S. Pi t t endr igh  
D e p a r t m e n t  of B i o l o g y  
Pr ince ton  Univers i ty  
P.O. B o x  704 
P r ince ton ,  New Jersey 
D r .  Allan 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 Minnesota 
Minneapolis,  Minnesota 
D r .  E. C. Pol lard 
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 .  Norman 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 C a l i f o r n i a  
B e r k e l e y  4 ,  C a l i f o r n i a  
D r ,  Sidney Fox 
Florida S t a t e  U n i v e r s i t y  
Tallahassee, Florida 
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C o p y  N o .  
33 
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36 
37 
38 
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R e c i D i e n t  
D r .  C a r l  Sagan 
H a r v a r d  C o l l e g e  O b s e r v a t o r y  
C a m b r i d g e  3 8  Massachusetts 
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  
National 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  
D r ,  D a l e  Jenkins 
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. 
D r .  R ,  E ,  C a m e r o n  
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  
D r ,  C a r l  B r u c h  
O f f  ice of Space Sciences  
National 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 ,  
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  
I I T  R e s e a r c h  I n s t i t u t e  
E d i t o r s ,  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, Feb. 15 - May 15, 1966

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