Analysis of vacuum effects in sterilization of microorganisms for planetary quarantine application

Brannen, J. P.

English

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Mcirca August 1970
Preprint. To be published in the German jotwnej Diophysik (Berlin,)
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An Analysis of Vacuum Effects
in the Sterilization of Microorganismst
J. P. Brannen
Planetary Quarantine Department
Sandia Laboratories
Albuquerque, New 11exico, USA
ABSTRACT
By using the thermodynamic formulation of the
absoluce reaction-rate theor y, variations in pressure
can be incorporated in a rational modal for dry-heat
sterilization of microorganisms. Coupling this model
to observations, a L-VT value of 15 to 65 liters/mole
is estimated for the sterilizing reaction. The theo-
retical AW" for D_XA is about 7. liters/mole_
O1n.ItrrVm tY
6MlMME SCIENCES CCV)IUNICAM MJEO
WAAIULNT Of WTEORAt AND FURLIC WAIN
'	 E6 ^ ^ 5 h 'L
	 THE OEOM WASNINOON UNIVE"ITY
tThis work was conducted under Contract No: W-3.2, 853, NASA Headquarters,
Washington, D. C.
1. Introduction
The effects of vacuum on the sterilization of microorganisms by heat is of
interest for planetary quarantine applications. It is not reasonable to assume
that the 10 17 torr pressure of outer space can be attained in a terrestrial
laboratory for the purpose of observing the effect on microorganisms. However,
ft is possible to carry out an analysis based on- . observations at greater pres-
sures, which provides the parameters necessary for gaining some insight into
the combined effect of vacuum and heat on microorganisms in outer space. In
addition, evidence is presented that the sterilization mechanism for B. subtilis=
by heat is a monomolecular reaction on one or two vital macromolecules. This
provides additional evidence to that which we . provided (Brennen, to be published)
for a thesis by liar (1963), that in fhe sterilization of B. subtilis by heat, we
are observing the action of heat on ANA in vivo.
2. Analysis and Model
The basic model for this work is the kinetic model discussed by this author
(Brennen, 1968). The basic assumption is that, in a dry-heat environment, ster-
ilization results from chemical reactions inside the microorganisms, and these
reactions "in the smaW have of-der. The Arrhenius formulation
k= - A exP (_EIT)
was used for the required reaction-rate constants. To investigate the effect of
pressure, we shall use the thermodynamic formulation of the absolute reaction-
rate theory. Here, kr, the reaction-rate constant, is given by
kr'T"p-
-2-
where h = Planck's constant, k - Boltzmann's constant, T is the temperature in
degrees Kelvin, R is the gas constant, and AF* is the Gibbs free energy.
i
A more complete formulation for AF' is
AF$ = AHD - TASD + pAVr
where AH"' is the activation enthalpy and ASO is the activation entropy, both at
!!t
	 • ..
0 pressure, Where p is the pressure and AV is the volume change as the reactant
goes into the activated state.
♦ 	 j	 ,
Usually, it is sufficient to set AVT = 0, which effectively includes the
pressure effect on the reaction-rate constant in AH$ and AS*. However, data
from Dairs, Silverman & Keller (1962) suggests that, in some eases at least,
the pAgT term should not be neglected. For example, they report that, under a
pressure of 10 8 torr and 60°C, only 40% of a population of B. subtilis sur-
viva after 5 days. This translates to 90% destruction in 12.5 days. On the
i	 j•
othar hand, Silverman (1966) reported a Alf" of 34.6 kcal/mole and a AST of
14.65 e.u. for the same organism under what one can presume to be a pressure
of 1 atmosphere. Assuming, as - did Silverman, 1 molecule being inactivated by
a first-order reaction as the sterilization mechanism, and using his values for
AN and AS$, 5t follows that 123 days should be required for the 90% reduction
at 60°C. In.the same report. Silverman gives AHr = 25,000 cal/mole and AS
21.1 e.u. for data generated by Xoesterer (1964). Using these values for AH*
and AS* -leads to a value of 24.8 days for 90% destruction at 60% and 1 at-
mosphere.	 L._ :
...Fro® a 60°C. 1-atmosphere, 90% reduction value (D value), and a 60°C
10 8 torn D value, reaction-rat . constants can be calculated by the formula
k  = -loge (0.1)/D .
An estimate for AV can now be obtained by means of the formula
AV* 8	 RT p loge (kp kp	 (1)
2	 1	 tt^ l^ 2^
where pl and p2 are pressures in atm, R is the gas constant in cc-atm, and
k and k are the reaction-rate constants at pressures p l and02 (Johnson,
P1	P2
Eyring Polissar, 1954, p. 305).
In this manner it was found that, with AR T = 34.6 kcal/moll and AS $ g
14.63 e.u.,
AV = 62,480.7 cc/mole
while, if AR# $ 25 kcal/mole and AS _ -11.1 e.u.,
AV = 18,770.1 cc/mole
Additional analysis without AR*'a and AS 'a can be carried out via equa-
tion (1), data presented by Silverman, Davis, and Keller (1964) and the re-
lationship
- - - log Ik	 = log D
	
(2)e PS 2	 e pp2
where Dp denotes D value at pressure p.
Silverman et al. report that after 5 days at 60'C, they found 72% survival
for B. subtilis at 1 atm and 40% survival at 16-6 torr. Employing D values
implied by these - data in equation (1) and the equation (2) relationship yields
AV# a 28 . 1 liters /mole• They also report that after 5 days .at 88°C, they found
0.012 B. subti lis survival at 10-6 torr and, at 90°C, they found 0.552 survival
In one experiment and 0 . 09X survival in another. Assuming no great difference
between 88% and 90% as regards survival, these results give AV # = 62 liters/
mole for the first experiment and 19 . 4 liters /mole for the second.
The AH$ and AS$ values computed by Silverman for his and Koesterer's data
roughly represent the extremes which one sees for B. subtilis. This plus the
Silverman, Davis, and Keller results . leads to the conclusion that AV# probably
dies between 15 and 65 liters/mole.
It was .reported by Silverman, Davis, and Keller that after 5 days at 25°C!
there were 1132 B. subtilis survivors under ultrahigh vacuum. This leads us*
to conclude that the vacuum effect alone is negligible and that it is the
kinetic effect of beat plus vacuum which leads to increased sterilization rates.
• The positive value for AV$ -ipdicates that the activated state of the 'vital
solecule" is less ionized than the unactive state. Since AV $ is generally posi-
tivz for unimolecular reactions (Johnson , •Eyring & Polissar, 1954, p. 306), the
positivity - of the AV$ computed from the Silverman-Koesterer data is consistent
with the assumption of a 1-molecule sterilization mechanism.
Assuming a unimolecular sterilization reaction, it is possible to approxi-
mate AV# for a very wide range of pressures by the formula
a
AV* R	
0.lb4V
b+r^+r2+1•'	 -
-5--
where b$ is the length of the bond that is broken, E b is the sum of the bond
lengths in the chain, V is the molar volume and r  and r2 are the covalent
radii of the terminal atoms (Glasstone, Laidler & Eyring, 1941, p. 473 10. This
formula in based on the-assumption that the volume change from reactant to acti-
vated state is all due to length changes ...id that the molecular cross section
remains constant.
Suppose the "vital" molecule responsible for sterilization is DNA. To com-
pare a AVM estimate for bond breakage in DINA with the AV T computed from the
Silverman-Koesterer data, we. will assume DNA has 20,000 nucleotide pairs (White,
Handler & Smith, 1964, p. 173), a molecular weight of 2.5 x 10 (Watson, 1955,
p. 85), bond lengths of 3 1 (Pauling, 1960, p. 503), and a specific volume of
0.55 mljg (Katz & Schachman, 1955).
Under these nssumption.s, the value of AVT for DNA is approximately 7 liters/
mole. This is in fair agreement with the AV# 'computed from Koesterer's data,
especially in view of the essumption that AV  results solely from length changes.
Since the volume of a mole of DNA is about 106 liters, these Otte represent a
volume change of approximately 0.003X.
-6-
3. Applications
z
Having an approximation fur AVT, pressure is easily incorporated as an
environmental parameter in a kinetic sterilization model. One must be care-
ful, however, in using the formula AF T/RT = (AHT - TASK
 + pAV^ VRT. Since
AV$ is in cc/deg mole, AST in cal/deg mole, and AH' in cal/mole, the gas con-
stant R used with AH$ and AST
 should be in cal/deg mole, while with AVM , it
Should be in cc atm/mole.
1
It follows that the AHT
 and AS values computed by Silverman, from the
point of view expressed here, actually include pAV' After AV has been de-
termined, it is possible to compute corrected values for-AH# assuming AST = 	 .
AS 	 For the Silverman data,
i
AHD = 33.081 kcal/mole
and, for the Roesterer data,
ART - 24.545 kcal/mole
Using these corrected values for AH and the p7eviously determined AV 8,
one is in a position to examine D values as a function of pressure. Table 1
shows comparison of D values computed for Silverman's and Koesterer's data at
a temperature of 125°C and for pressures from 1.3 x 10 -11 to 4 atm. (It was
found in the computation that, with the available computing equipment, a
pressure of 1.3 x 16-11 atm - 10 8 torr and 0 atm were indistinguishable.)
It is also of interest to examine D values at a low pressure as a function
of temperature. Table 2 shows a comparison of calculated D values for Silverman's
and Koesterer • s data as a function of temperature at 1.3 x 10 -11 atm.
4. Conclusions
The primary purpose of this paper is the presentation of a model and an
analysis technique for considering pressure as an environmental parameter in
dry-;.eat sterilization. The outcome of such calculations is entirely de-
F-ndent on the input data, and the data used for this analysis were not gen-
erated with this analysis in mind. Consequently, one should hesitate to draw
'	 1
firm conclusions from this analysis. However, the AV' value of 7 1/mole for
DNA is within an order of magnitude of the-15- to 65-1/mole values computed
for Silverman's and %oesterer's observations with - B. subtilis. Under these
conditions, the agreement is good. The fact that the lower AVM value was
obtained for DNA raises the question of what else in the microbe could lead
to a greater AVM
 value. We then. are led to agree with Wax that, in the
sterilization of B. subtilis by heat, we are likely observing effects on DNAJ
"	 E
in vivo.
The input data for this analysis were dry-heat sterilization data. From
these, it followed that D values increased with pressure. Within limits, it
hits been found that this is true in solutions under hydrostatic pressure
(Johnson b Zobell, 1949).
An examination of Table 2 suggests that a si.;nificant decrease in a space-
craft's bioburden can occur in outer space, provided the temperature can be
maintained at above 50°C.
-8-
TABLE 1	 .
Calculated 125°C D values as a function of pressure.
It is assumed all other eaviroamental parameters are
constant.
Pressure	 D values in minutes, based on
(atm)	 Silverman's data	 Koesterer's data
	
1.3 X 1011 	 4.3	 35.5
	
0.2	 6.3	 40.2
	
0.4	 9.2	 45.5
	
0.6	 13.5	 51.4
	
0.8
	
19.8	 58.2
	
1.0
	
29.0	 65.8
	
1.4
	
62.3	 84.3
	
2.0	 196.4	 122.0
	2.4	 422.3	 156.1 .
3.0 . 	1330.7	 226.1
	
3.4	 2860.3	 289.3
	
4.0	 °014.1	 418.9
-9-
• TABLE 2
D values calculated for a pressure of 1.3 X 10-11 atm = 10-8
 torr
_	 as a function of temperature.	 Its assumed all o*her environ-
mental parameters are constant.
Temperature D values in jays, based on
(deg C) Tilyerman's data Koesterer's data
0 902,643.0 52,327.0
10 100,920.0 10,227.0
20 13,088.0 2,232.0
30 1,940.0 538.0
40 -	 324.6 142.0
50 60.6	 - 40.6
60 -	 12.5 12-5
70 2.8 -4.1
80 0.69 1.45
9.0 0.18 0.537
100 0.052 0.21
_10-
Brannen, J. P. J. Theoret. Biol., In press.
Brannen, J. P. (1968). Math. Biosci., 2, 165-179.
Davis, N. S., Silverman, G. J. b Keller, W. H. (1962). ARpl. Microbiol., 11,
202-210.
Glasstone, S., Laidler, K. J. 5 Eyring, H. (1941). "The Theory o:: Rate
Processes," McGraw-Hill, New York.
Johnson, F., Eyring, H. b Polissar (1954). "The Kinetic Basis of Molecular
Biology," Wiley, London.
Johnson, F. H.	 Zobell, C. E. (1949). J. of Bact., 57, 353-358.-
Katz, S. & Schachmarr, H. K. (1955). Biochemica et Biophysica Acta, 18, 28-35.
Koesterer, M. G. (1964). Develop. Ind. Microbiol., 6, 268-276.
Pauling, L. (1960). "The ;Nature of the Chemical Bond," Cornell Univ. Press,
Ithaca.
Silverman, G. J. (1966). The - Resistivity of Microorganisms to Therrial In-
activation by Dry Heat, NASA-CR-70029. Report prepaied under NASA Contract
RSG--691.
Silverman, G. J., Davis, N. S. 6 Keller, W. H. (1954). In "Life Sciences and
Space Research" (Florkin, M. and Dollfus, A., eds), Wiley, New York.
Watson. J. D. (1955). "Molecular Biology of the Gene," W. A. Benjamin; New York.
Wax, R. G. (1963). "Thermal Inactivation of Spares of a Thv^"ine Requiring St,;raim
of Bacillus subtilis,• Ph.D Z$esis, Penn. State.Univ.
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An analysis of vacuum effects in the sterilization of microorganisms

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