Bacillus spore germination in simulated Martian environmen

Hagen, C. A.

English

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I 
R e p o r t  N o .  I I T R I - L 6 0 2 3 - 4  
( 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  I N  E X T R A T E R R E S T R I a  ENVIRONMENTS 
Contract N o .  N A S r - 2 2  
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  
I I T  R E S E A R C H  I N S T I T U T E  
Report N o ,  IITRI-L6023-4 
(Quar t e r ly  S t a t u s  Report) 
LIFE I N  EXTRATERRESTRIAL, ENVIRONMENTS 
November 15,  1965 to  February 15,  1966 
National Aeronautics and Space Administration 
Contract N o ,  NASr-22 
I I T R I  P ro jec t  L6023 
I , INTRODUCTION 
I t  has been established tha t  spores of a s t r a i n  of 
Bac i l lu s  cereus ,  isolated f r o m  Cal i forn ia  desert s o i l  
germinate and grow vege ta t ive ly  i n  the  absence of oxygen 
i n  a simulated Martian environment w i t h  d i u r n a l  temperature 
cycles (-65 t o  25OC) and 8% so i l  moisture ,  I t  has  a l s o  been 
e s t a b l i s h e d  that  Bo s u b t i l i s  spores no t  germinating anaero- 
b i c a l l y  germinate and grow vegeta t ive ly  wi th  subsequent 
spo ru la t ion  when the p a r t i a l  p ressure  of oxygen i s  10 mm 
of H g  i n  the same environment, B o  cereus r e q u i r e s  an oxygen 
pa r t i a l  pressure  of 15 mm of Hg f o r  spo ru la t ion ,  
Determination of the  effect  of 8-, 16- ,  and 20-hr d i u r n a l  
f r e e z e  cyc le s  on t h e  growth response of B o  cereus  and 
Bo s u b t i l i s  spores during 56 d a y s  i n  a simulated Martian 
environment modified by 8 t o  lP/O s o i l  moisture and an oxygen 
p a r t i a l  p re s su re  of 15 mm of Hg w a s  completed dur ing  the  past 
I I T  R E S E A R C H  I N S T I T U T E  
1 
year ,  Deta i led  d a t a  a r e  repor ted  i n  Reports N o ,  IITRI-L6023-1, 
-2 ,  and -3, I n  summary, w e  found tha t :  
(1) Extension of the  f reeze  cyc le  from 8 t o  16 h r  
delayed Bo cereus spore g e m i n a t i o n  and subse- 
quent  vege ta t ive  eel1 growth 2 days and sporu- 
l a t i o n  a t  least  4 days,  Bo s u b t i l i s  spore 
germination, vege ta t ive  ce l l  growth, and sporu- 
l a t i o n  w e r e  delayed a t  l e a s t  2 days,  
( 2 )  Extension of the  f r eeze  cyc le  from 8 t o  20 h r  
delayed Be cereus spore g e m i n a t i o n  and vege- 
t a t i v e  ce l l  growth 6 days,  Bo s u b t i l i s  spore  
germination and vegeta t ive  ce l l  growth w e r e  de- 
layed a t  least  3 days, Sporulat ion of n e i t h e r  
organism w a s  apparent during 56 days,  
(3)  The growth response (vege ta t ive  cel l  growth 
and sporu la t ion)  of B,  s u b t i l i s  w a s  more r ap id ,  
wi th  maximum populat ions reached before  those 
of Bo cereus ,  
(4) After  56 days i n  the  simulated X a r t i s n  envf rnn-  
men t ,  g r e a t e r  than 50% of the  v i ab le  B o  cereus 
cells  w e r e  spores  except those subjec ted  t o  20-hr 
f r eeze  cyc le ,  L e s s  than 50% of t h e  v i ab le  
B e  s u b t i l i s  cells  w e r e  spores ,  
I I T  R E S E A R C H  I N S T I T U T E  
2 
I .  
1 
Bo cereus and Bo subtilis spores produced in 
the simulated Martian environment retained 
their viability and were able to reestablish 
an ecological niche when transferred into a 
similar environment, 
Thermal-death time studies with Bo subtilis 
indicated that no change in thermal resistance 
occurred in spores produced in the simulated 
Martian environment, Similar studies with 
Bo cereus were not conclusive because of 
initially low spore populations, 
Dry-heat studies with B o  cereus spores in 
simulated Martian soil indicated that ex- 
posure to either 100 or 130OC for as long 
as 30 min was not sufficient to sensitize 
the spores to a simulated Martian environ- 
ment with diurnal freezing and thawing cycles, 
Although spore germination, vegetative cell 
growth, and sporulation were delayed pp- 
lation densities after 28 days of exposure to 
the environment were the same as those of un- 
heated spores 
I I T  R E S E A R C H  I N S T I T U T E  
/ 
Other s t u d i e s  w e r e  concerned with possible anaerobic 
sporu la t ion  of B o  cereus  and vegeta t ive  cel l  growth and 
sporu la t ion  of B, s u b t i l i s  i n  the simulated Martian environ- 
ment, wi th  2 ,  0,2, and 0,02% potassium n i t r a t e  as  a terminal  
e l e c t r o n  acceptor  i n  place of oxygen, Possible vege ta t ive  
ce l l  growth of B o  cereus occurred a t  0,02% n i t r a t e  concen- 
t r a t i o n ,  while B o  ~~ s u b t i l i s  w a s  able t o  grow vege ta t ive ly  a t  
a l l  three concent ra t ions ,  Sporulation of ne i the r  o rgan i sm 
occurred 
S tudies  during the p a s t  qua r t e r  w e r e  concerned w i t h  the 
growth response of B ,ce reus  and -__ B o  s u b t i l i s  spores  i n  a 
simulated Martian environment with l o w  barometric p re s su res  
between 5 and 1 2  mm of H g ,  Preliminary s t u d i e s  ind ica t ed  
t h a t  the growth response of these o rgan i sms  w a s  unusual ly  
delayed o r  i n h i b i t e d  by t h e  l o w  barometric p re s su re ,  Fur- 
ther s t u d i e s  showed t h a t  s o i l  p a r t i c l e  s i z e  and/or p H  are 
con t r ibu t ing  f a c t o r s ;  t he re fo re  f u r t h e r  w o r k  i s  required 
before conclusions concerning the e f f e c t s  of barometric pres- 
s u r e  a re  drawn, 
I I EXPERIMENTAL PROCEDURES 
T h e  Bac i l lu s  spec ie s  used were the I I T R I  s t r a i n  of B, 
s u b t i l i s  and a B o  cereus  s t r a i n  i s o l a t e d  from Ca l i fo rn ia  
I I T  R E S E A R C H  I N S T I T U T E  
4 
desert s o i l .  The spore  suspensions,  prepared i n  the  manner 
descr ibed i n  Reports N o ,  I ITRI-6194-12  and -13, w e r e  hea t -  
shocked f o r  10 m i n  a t  8OoC immediately before  use ,  
Experiments i nves t iga t ing  the  e f f e c t  of barometric pres -  
sure  on the  growth response of Bo cereus and B o  s u b t i l i s  
w e r e  conducted under the  following environmental condi t ions :  
(1) Daily f r eeze  cyc le  of 8 h r  
( 2 )  Fe l s i t e / l imon i t e  s o i l  containing 1% organic  
medium 
( 3 )  Moisture concent ra t ions  of 7 ,  9 ,  and 10% 
(4) Barometric pressures  of 5 ,  9 ,  1 2 ,  and  75 mm 
of Hg, 
Water i s  i n  t he  vapor s t a t e  a t  2506 under barometric pres -  
su res  of 5 ,  9 ,  and 1 2  mm of Hg, The moisture  i n  the tubes was 
f rozen  i n  a dry ice-acetone ba th  p r i o r  t o  es tabl ishment  of 
p re s su re ,  Freezing was conducted t o  maintain the  moisture a t  
a cons t an t  l e v e l  although we r ea l i zed  t h a t  when t h e  f rozen  
mois ture  i n  t he  tubes thaws, the water vapor con t r ibu te s  t o  
the  p re s su re  of t h e  system i n  accordance wi th  Da i ton ; s  law 
of p a r t i a l  p re s su res  
The pH of the simulated Martian s o i l  was ad jus t ed  by a l -  
t e r i n g  t h e  r a t i o  of l imoni te  t o  f e l s i t e ;  l imon i t e  is  a c i d i c  
and f e l s i t e  i s  a l k a l i n e ,  
I I T  R E S E A R C H  I N S T I T U T E  
5 
1 
I The par t ic le  s i z e  of the simulated Martian s o i l  was ad jus ted  
by a l t e r i n g  the  r e l a t i v e  amounts of p a r t i c l e s  i n  t h e  fol lowing I 
I s i z e  ranges: 590 t o  297 p ,  297 
105 p ,  105 t o  74 p 9  7 4  to  44 p ,  
Tota l  and spore counts w e r e  
I 
I 
I 
t o  210 p ,  210 t o  149 p ,  149 t o  
and < 44 p e  
performed i n  a l l  s t u d i e s  a t  0 ,  
I 
1, 2 ,  7 ,  and 28 days un le s s  otherwise s t a t e d ,  T h e  recovery medium 
I 
I was trypticase soy agar  (BBL) Be cereus  was incubated f o r  24 hr 
I a t  35OC, and B, s u b t i l i s  was incubated f o r  48  h r  a t  35OC, 
I 111. RESULTS AND DISCUSSION 
Figures  1 and 2 appeared i n  Report N o ,  IITRI-L6023-3 and are 
shown he re  t o  in t roduce  the problem encountered during t h i s  quar- 
ter. A t  t h e  t i m e  t he  pre l iminary  r e s u l t s  w e r e  obtained i t  d id  
no t  s e e m  unusual t h a t  the  growth responses of Be cereus and 
Be s u b t i l i s  had been severe ly  repressed s i n c e ,  we  reasoned, a 
lowering of t h e  barometric pressure e f f e c t s  t he  water a v a i l a b l e  
t o  a microorganism, Water i s  i n  t he  vapor s t a t e  above 2 and 1 4 O C  
a t  barometr ic  p re s su res  of 5 and 1 2  mm ot Hg, r e spec t ive ly ,  I f  
l i q u i d  w a t e r  w e r e  not  a v a i l a b l e  to  the organism, t h e  growth cyc le  
would be confined t o  s h o r t  t imeper iods  dur ing  f r eez ing  and thawing 
i n  t h a t  region below t h e  temperature maxima of 2 o r  1 4 O C ,  
l l T  R E S E A R C H  I N S T I T U T E  
6 
E60 23  
February 18, lQ66 
Figure 1 
E F F E C T  OF BAROMETRIC PRESSURE ON THE GROWTH RESPONSE 
O F  BACILLUS CEREUS I N  A SIMULATED MARTIAN ENVIRONMENT 
E x p e ~  m e r , t a l  C o n d i t i o n s :  N o  f l u s h i n g  of t u b e s  p r l o r  t:o s e a l i r l q ,  
7% moi s tu re ,  d i u r n a l  t e m p e r a t u r e  cyc l  e 
( - 6 5  to 2 5 ' C )  with 8 h r  Ererze cyclt3. 
I- 
I 
I 
7 1 2 
25 
7 
Figure 2 
10 
10 
4 10 
10 
E F F E C T  O F  BAROMETRIC PRESSURE ON THE GROWTH RESPONSE 
OF BACILLUS S U B T I L I S  I N  A SIMULATED MARTIAN ENVIRONMENT 
Experimental Conditions: No flushing of tubes prior to sealing, 9% moisture, diurnal temperature cycle 
(-65 to 25OC)  with 8 hr freeze cycle. 
75 mm Hg barometric pressure, total count 
75 mm Hg barometric pressure, spore count 
12 mm Hg barometric pressure, total count 
12 mrn Hg barometric pressure, spore count 
O--a  
A-i 
&--A 
0 
/ 
/ 
1 2 7 2 R  0 
However, e x p e r i m e n t s  wi th  environmental cond i t ions  t h a t  
should have permit ted growth of Bo cereus  w e r e  nega t ive ,  The 
following f a c t o r s  w e r e  considered: 
(1) Change i n  t he  growth response of t h e  b a c t e r i a l  
c u l t u r e s  
( 2 )  Improper s o i l  subs t r a t e  
( 3 )  Too l i t t l e  a v a i l a b l e  water 
( 4 )  Absence of organic  medium, 
While checking the  above f a c t o r s  w e  l earned  t h a t  a new 
f e l s i t e / l i m o n i t e  soil with  a pH of 8 , 7  t o  8,8 had been 
r e c e n t l y  prepared,  Maximum i n i t i a t i o n  of spore germination 
usua l ly  occurs  between pH 6 and 8 ,  but  the  optimum pH may 
vary wi th  the  i n i t i a t i n g  substance,  Lawrence ( r e f ,  1) found 
an optimum near pH 8 f o r  Bo c e r e u s ,  Vas and P r o s z t  ( r e f ,  2 )  
found t h a t  below pH 6 the  i n i t i a t i o n  of € 3 % ~ ~ u s  spores f e l l  
s l i g h t l y  but  decreased suddenly a t  pH 5,  
( r e f ,  3 )  obtained more i n i t i a t i o n  of B ,  s u b t i l i s  spores  a t  
pH 8 , 5  than a t  pH 7 o r  6 ,  However, Wolfe and Thorley ( r e f ,  4) 
found that, more germination was i n i t i a t e d  wi th  B o  s u b t i l i s  
spores  by glucose a t  pH 5 ,5  than a t  pH 7 ,5  o r  8 ,5 ,  bu t  with 
L-alanine more i n i t i a t i o n  occurred a t  8 ,5 ,  
Wolfe and Mahmoud 
-__-_- 
Therefore the  optimal pH for  i n i t i a t i o n  of spore germr- 
na t ion  appears t o  be dependent upon the  b a c t e r i a l  s t r a i n  
and t h e  s u b s t r a t e ,  The pH of 8 ,7  t o  8 ,8  found i n  our 
IIT R E S E A R C H  I N S T I T U T E  
9 
I 
experiments i s  a l i m i t i n g  value f o r  optimal spore germination 
of Bo cereus and Bo s u b t i l i s ,  
Figure 3 shows the  growth response of d i f f e r e n t  s tock  spore 
suspensions of B o  cereus i n  t he  a lka l ine  s o i l  and of one such 
suspension i n  s o i l  with the  pH adjusted t o  7,2 by the  addi t ion  
of more l imonite ,  The two spore suspensions inoculated i n t o  
the  more a l k a l i n e  soil did not  germinate during the 7 and 2 8  
days of t he  experiment, The spore suspension ifioculated into 
the  soil.  wi th  a pH of 7,2 d id  germinate, wi th  subsequent vege- 
t a t i v e  ce l l  growth occurr ing between 2 and 7 days,  N o  sporu- 
l a t i o n  occurred with t h i s  c u l t u r e ,  and w e  be l ieve  t h a t  t h i s  
f ind ing  i s  r e l a t e d  t o  one o r  more of the  fol lowing f ac to r s :  
(1) S ize  of soil p a r t i c l e s  
( 2 )  pH of so i l  
( 3 )  Available s o i l  moisture,  
Pre l iminary  conclusions ind ica t e  t h a t  increas ing  t h e  l imoni te  
f r a c t i o n  a f f e c t s  t he  amount of bound w a t e r ,  Limonite i s  r e a d i l y  
hydrated t o  g o e t h i t e  and competes wi th  the  microorganisms f o r  
a v a i l a b l e  water ,  T h i s  and “Lliie o the r  f a c t o r s  above a r e  being 
i n v e s t i g a t e d ,  
I I T  R E S E A R C H  I N S T I T U T E  
10 
F i g u r e  3 
1 c4 
'1 
- 
1 I I I 
THE EFFECT OF p H  AND SOIL TYPE ON BACILLUS CEREUS SPORE GERMINATION 
I N  A S W L A T D  MARTIAN ENVIRONMENT 
Experimental  Condi t ions :  D i u r n a l  tempera ture  c y c l e  (-65 t o  7.50'2) 
w i t h  8-hr f r e e z e  c y c l e ;  15-nun Hg oxygen 
p r e s s u r e .  
/ 
T o t a l  count: s p o r e  suspens ion  p r e p a r e d  8-26-65; fe ls i t r / l  
soil, pH 8.4; 10% m o i s t u r e  
o--o Spore  count: s p o r e  suspens ion  p r e p a r e d  8-26-65: f e l s i t e / l  
soil, pH 8.4: lO"6 m o i s t u r e  
T o t a l  count ;  s p o r e  suspens ion  prepared  11-11-65: f e l s i t e /  
soil, p H  8.4: 1PL m o i s t u r e  
b.-- 4 Spore count; spore  suspens ion  p r e p a r e d  11-11-65: f e l s i t e / l i m o n i t e  
I)----c1 T o t a l  count: s p o r e  suspens ion  p r e p a r e d  11-11-65: f e l s i t e / l i m o n l t e  
[I - - -0 Spore count: s p o r e  suspens ion  p r e p a r e d  11-11-65; f e l s i t e / l i n r a n i t e  
so i l ,  pH 8.4; 10% m o i s t u r e  
s o i l ,  p H  7.2: 10% m o i s t u r e  
soil, pH 7.2: 10% m o i s t u r e  
-0 T o t a l  count: spore suspens ion  
so i l ,  pH 7.0: 7% mois ture  
p r e p a r e d  8-26-65: f e l s i t e / l i m o n i t e  
0- - a s p o r e  count: s p o r e  suspens ion  p r e p a r e d  8-26-65: f e l s i t e / l i m o n i t e  
soil, p H  7.0; 7x m o i s t u r e  
I 
I V ,  SUMMARY ~ _ _ _ - -  
A summary of the  r e s u l t s  from the p a s t  year  w e r e  presented 
i n  Sect ion I ,  T h i s  d a t a  showed t h a t  Bo - cereus  spores  g e m i n a t e  
wi th  subsequeEt vege ta t ive  ce l l  growth i n  a simulated Martian 
environment devoid of oxygen but  they requi red  oxygen f o r  
spo ru la t ion ,  
R e c e n t  da t a  i n d i c a t e  t h a t  B o  cereus and - -  Bo s u b t i l i s  - - ~ -  can  sus- _._s-__l 
t a i n  an eco log ica l  niche in a simulated Martian environment modi- 
f i e d  by 8 t o  loo /o  moisture and an oxygen p a r t i a l  p re s su re  of 
15 mm of Hg, 
The Martian environment was not l e t h a l  t o  hea t - t r ea t ed  spores  
of E, cereus ,  
The i n h i b i t i o n  of _---- Bo cereus and B, s u b t i l i s  spore germination 
by reduced barometric p re s su res  s imulat ing recent e s t ima tes  of 
the environment of Mars i s  being reexamined i n  t e r m s  of s o i l  pH 
and p a r t i c l e  s i z e ,  
V, PERSGNNEL AND RECORDS 
E x p e r i m e n t s  w e r e  planned with t h e  counsel of D r ,  E ,  J c  
Hawrylewicz and with the  technica l  a s s i s t ance  of M i s s  Marjorie 
Ewing and M i s s  Vivian Tolkacz, 
I I T  R E S E A R C H  I N S T I T U T E  
1 2  
E x p e r i m e n t a l  data  are recorded i n  I I T R I  L o g b o o k s  C 1 6 3 9 4 ,  
C l 6 4 1 3 ,  and C 1 6 5 9 0 ,  
R e s p e c t f u l l y  s u b m i t t e d  
I I T  RESEAFtCH INSTITUTE 
C h a r l e s  A, H a g e n  
A s s o c i a t e  B a c t e r i d l o g i s t  
L i f e  Sciences R e s e a r c h  
A p p r o v e d  by : 
L i f e  Sciences R e s e a r c h  
l l T  R E S E A R C H  I N S T I T U T E  
13 
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20 
30 
4 0  
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Space Sciences Laboratory 
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O f f i c e  of Space Sciences 
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16 


Life in Extraterrestrial Environments Quarterly Status Report No. 4, 15 Nov. 1965 - 15 Feb. 1966

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