articulo estabilidad sorokulova-2008
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ARTICLES: FORMULATION ANDENGINEERING OF BIOMATERIALS
Novel Methods for Storage Stability and Release of Bacillus Spores
Iryna B. Sorokulova, April A. Krumnow, and Suram PathiranaDept. of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849
Arnold J. MandellDept. of Neuroscience, Psychiatry and Behavioral Sciences, Emory School of Medicine, Atlanta, GA 30322
Dept. of Psychiatry, School of Medicine, UCSD, La Jolla, CA 92037
Dept. of Mathematical Science, FAU, Boca Raton, FL 33431
Vitaly VodyanoyDept. of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849
DOI 10.1021/bp.22 Published online September 17, 2008 in Wiley InterScience (www.interscience.wiley.com).
Bacillus subtilis spores were immobilized in activated charcoal and tapioca and filled with acacia gum. These formulations were tested for spore stability during storage at tem-
peratures ranging from 408C to 908C and for bacterial release. Thermodynamic analysisshowed that immobilization of spores in acacia gum significantly increased their viabilitycompared with unprotected spores. The viability was further increased when suspensions of spores in acacia gum were added to granules of charcoal and tapioca. The number of thespores released after storage was also increased when spores were treated with acacia gum
prior to immobilization in tapioca and charcoal. Formulations of Bacillus spores with acaciagum and porous carriers (charcoal and tapioca) prolong the anticipated shelf-life of sporeseven under ambient temperature and provide slow and steady bacterial release consistent with their high viability.
Keywords: acacia gum, shelf life, Arrhenius and Eyring equations, Gibbs free energy,enthalpy-entropy compensation
Introduction
Bacteria of the Bacillus genus are usually considered to
be very stable due to their formation of spores. The experi-
mental data,1 however, indicate that only a few individual
Bacillus spores from an initially large population can sur-
vive for years. In particular situations, it is important to
store Bacillus spores without significant changes in their
titer during long-term storage. For example, bacteria of the
Bacillus genus are widely used as highly effective pro-
ducers of antibiotics, enzymes, aminoacids, and vitamins.2
Various Bacillus strains are known for their roles as probi-
otics for humans and animals, biopesticides, bioinsecticides,
and biological indicators.2,3 Such strains-producers require
special methods of storage to maintain a stable level of
spore viability. Some of them, such as the biopesticide
strains, should be formulated in such a way as to ensure the
slow release of spores.4 To avoid a decrease in their
numbers, Bacillus spores are frequently frozen, freeze-
dried, or refrigerated in aqueous suspensions or buffers.5
Even under these conditions, some Bacillus spores show a
significant loss in viability.6 Numerous formulations that
make use of a variety of polymers have been used to pro-
long the shelf life of spores.7
The two aims of this study are (1) to explore the effective-
ness of preserving of Bacillus spores in new formulations
using charcoal and tapioca as carriers along with acacia gumpolymers to increase shelf-life and the slow and steady
release of spores, and (2) to use thermodynamic characteris-
tics of new formulations under various storage conditions for
the prediction of spores viability.
Materials and Methods
Bacterium
The bacterium used as an experimental model was type
strain Bacillus subtilis ATCC 6051 that was obtained from
American Type Culture Collection (Manassas, VA). B.Correspondence concerning this article should be addressed to V.
Vodyanoy at vodyavi@auburn.edu.
Biotechnol. Prog. 2008, 24, 1147À1153 1147
VVC 2008 American Institute of Chemical Engineers
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subtilis is regarded as a model Gram-positive organism for
genetic and physiological studies.8
Spore preparation
A fresh, overnight culture of B. subtilis ATCC 6051 was
spread-plated on Difco Sporulation Medium.9 The plates
were then incubated at 378C for 5 days. Sporulation was
assessed by phase contrast microscopy which revealed$99% phase bright spores. Spores were collected by flood-
ing the surface of the culture with sterile distilled water fol-
lowed by scraping with a sterile cell spreader (Daigger,
Vernon Hills, IL). Spores were then washed twice with ster-
ile distilled water followed by centrifugation at 19,837g for
40 min. To eliminate vegetative cells, they were heated at
808C for 20 min. Spores were then stored in sterile distilled
water at 48C until needed. Plates were incubated at 378C
overnight and the titer of spores was determined by plating
serial dilutions of spore suspension onto nutrient agar (NA)
plates.
Spore carriers and preparation of the protective biopolymer
Activated charcoal (Sigma, St. Louis, MO) and granu-
lated tapioca (Frontier, Norway, IA) were used as carriers
for B. subtilis spores. Acacia gum (Colloids Naturels
International, Rouen, France) was used as the protective
polymer. The solution was prepared by mixing the poly-
mer powder with sterile water in concentrations of 15%
(w/v). The solution was stirred and heated at 808C until
the powder was completely dissolved. It was then filtered
using two Brew Rite1 coffee filters (Rockline Industries,
Sheybogan, WI) in a Buchner funnel with vacuum filtra-
tion and autoclaved at 1208C, 20 PSI for 15 min, cooled
and stored at 48C.
Formulation of spores in the carriers and polymer
All the formulations were prepared using the Fluid Bed
System (Applied Chemical Technology, Florence, AL) at an
outlet temperature of 708C:
1. Tapioca þ spores, TþS: 75 mL of the spore suspension
was diluted with 300 mL of sterile water and dried with
375 g of tapioca.
2. Tapioca þ (spores þ acacia gum), TþSA: 75 mL of
the spore suspension was diluted with 300 mL of the 15%
solution of acacia gum and dried with 375 g of tapioca.
3. Charcoal þ spores, CþS: 75 mL of the spore suspen-
sion was diluted with 300 mL of sterile water and dried with375 g of charcoal.
4. (Charcoal þ spores) þ acacia gum, CCSþA: 375 g of
charcoal was mixed with 75 mL of the spore suspension.
The solution of acacia gum was prepared by adding 75 mL
of sterile water to 300 mL of the 15% (w/v) acacia gum
solution. The charcoal-spores preparation was then dried
with the acacia gum solution.
5. Charcoal þ (spores þ acacia gum), CCþSA: 75 mL of
the spore suspension was diluted with 300 mL of the 15%
solution of acacia gum and the mixture dried with 375 g of
charcoal.
Freshly prepared spores were used in all formulations.
The moisture content in prepared formulations was deter-
mined by Karl Fisher titration.10
Heat resistance test for spore viability
B. subtilis spores in the formulations described above
were tested for resistance to degradation at a range of
temperatures. The spore formulations were put in 50-mL
Falcon tubes (Becton Dickinson, Franklin Lakes, NJ), each
containing 500 mg of the formulation. Spores (S) and
spores with acacia gum (SþA), prepared in the same ratio
as in the formulations, and were used for the storage as a
control. Tubes with the varying formulations were placedin incubators at temperatures of: 40, 55, 60, 65, 75, 80, 85,
and 908C. Enumeration of viable spores was performed for
all formulations before placing for storage and at the pre-
determined time intervals during the storage. Five millili-
ters of cold sterile water were added to each tube and
mixed thoroughly. Samples containing charcoal and tapioca
were homogenized using the Tissue Tearor (model 985-
370; Biospec Products, Racine, WI). Decimal dilutions in
sterile water were prepared and appropriative dilutions
were plated onto NA (Difco, BD, Sparks, MD). Plates were
then incubated at 378C for 18–24 h. The number of spores
(colony forming units, CFU) was counted for each time
point and their mean and standard deviation were calculated
in triplicate or more.
A thermodynamic model of the thermal degradation
of spores
The apparent rate of degradation of spores during dehydra-
tion at constant temperature can be described by first-order
kinetics
dN
dt ¼ ÀkN (1)
where N is the concentration of spores being degraded, k represents the first-order rate constant, and t , the time of the
thermal drying. The solution of the equation (Eq. 1) can be
expressed as
N = N 0 ¼ eÀkt (2)
where N 0 is the initial concentration of spores and N / N 0 rep-
resents the ratio of the concentration of degraded spores to
the initial spore concentration. This fraction defines the ratio
of viable of spores. Taking the logarithm of both sides of
Eq. 2, one obtains
logð N = N 0Þ ¼ À2:303 Â kt (3)
A plot of the left-hand side of Eq. 3 vs. time (t ) yields an
estimate of k from the slope. The Arrhenius equation11 can
be used to express the temperature dependence of the first-order activation kinetics:
k ¼ Ae Ea RT (4)
where R is the universal gas constant, Ea represents the appa-
rent activation energy, and A is the pre-exponential Arrhe-
nius factor. Taking the logarithm of Eq. 4 yields
log k ¼ ÀEa
2:303 RÂ
1
T þ log A (5)
If the logarithm of k in Eq. 5 is plotted against the re-
ciprocal of temperature, 1/ T , then the slope of this graph
yields the activation energy ( Ea), the thermal activation
level of transitions from spore viability to nonviability.
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The r ate c onstant of the spore degra da tion proce ss
depends on the thermodynamic activation parameters of
this transition state and can be described by the Eyring
equation12:
k ¼k BT
he
ÀDG RT ¼
K BT
heDS R e
ÀD H RT (6)
where k is the rate constant, DG is the standard Gibbs freeenergy of activation, h is Planck’s constant, DS is the stand-
ard entropy of activation, D H is the standard enthalpy of
activation, k B is the Boltzmann constant, R is the gas con-
stant, and T is the absolute temperature in Kelvin. Taking
the logarithm of both sides of Eq. 6, one obtains
logk
T
¼ log
k B
h
þ
DS
2:303 RÀ
D H
2:303 RÂ
1
T (7)
If the plot of the left-hand side of Eq. 7 vs. 1 / T is linear,
one can compute the value of D H from the slope, DS from
the ordinate intercept and the Gibbs free energy of activation
by the relation:
DG ¼ D H À T DS (8)
From Eq. 4 we can define the energy of activation as
Ea ¼ À R@ ln k
@ 1T
À Á (9)
The substitution of k in Eq. 9 with the k equivalent relation
in Eq. 6 and differentiation of this new expression with
respect to 1/ T shows that the Arrhenius (Eq. 4) and the Eyr-
ing (Eq. 6) expressions can be related as
Ea ¼ D H þ RT (10)
and
A ¼ek BT
h
eDS R (11)
Taking the logarithm of both sides of Eq. 11, one obtains
log A ¼ logekT
h
þ
DS
2:303 R(12)
suggesting a linear relation between the logarithm of the
Arrhenius frequency factor and entropy of activation
obtained from the Eyring equation for the transitional state.
The first-order kinetic model (Eq. 3) was used to deter-
mine the best fits of the data with the apparent rate constant
k . The Arrhenius and Eyring equations were then applied to
the data in order to determine Ea, A, DG, D H , and DS of the
temperature-dependent viability transitions.
Enumeration of spore release
Each sample consisting of 1 g of the indicated spore for-
mulations was put in 50-mL Falcon tubes containing 10 mL
of sterile water, mixed and maintained at room temperature.
Before sampling for the determination of spore number, the
tubes were mixed again and 0.5 aliquots of supernatant were
removed at predetermined times. To maintain constant vol-
ume, 0.5 mL of sterile water was added to the tubes after
each sample was removed. Serial 10-fold dilutions of eachsample were prepared in sterile water and plated onto NA.
The plates were then incubated at 378C for 18–24 h. The
mean and standard deviation of the number of released
spores were calculated for at least triplicate determinations.
Results
Characterization of the formulations
The number of spores in the various formulations pro-
duced using the fluid bed system varied slightly, ranging
from 4.2 Â 108 to 7.9 Â 108 CFU/g. The moisture content
was low in all the formulations, varying from 0.21% Æ
0.01% in the products with tapioca to 1.4% Æ 0.06% inthose containing activated charcoal.
Heat resistance of spores on different carriers
The decrease in spore viability in time was dependent on
temperature and the presence or absence of the acacia gum
polymer and the carrier used. Figure 1 shows the typical de-
pendence of spore viability as a function of time at different
temperatures exemplified by the data obtained for spores that
were first suspended in acacia gum and then combined with
charcoal. The degradation rate followed first order kinetics
in time across almost all temperatures. The apparent rate
constants for spore degradation in the various formulations
were used to obtain linear Arrhenius and Eyring plots con-stants with Eqs. 5 and 7 and shown in the corresponding
Figures 2a,b. These plots were then utilized for the calcula-
tion of Ea, A, DG, D H , and DS. Those values are shown in
Table 1.
The estimated time for spores to lose one log of their ini-
tial titers at different temperatures was calculated using the
experimental values of the apparent rate constants of spore
degradation in k , 1/day for different formulations and the
Arrhenius equation (Figure 2c). Stability of spores with both
carriers was higher when acacia gum was added.
The highest viability was obtained for spores that were
absorbed by charcoal and filled with acacia gum (Table 1,
Figure 2d). In this formulation, after 2 years of storage at
208C, the predicted viability of spores is 90.5%. At this
Figure 1. Viability of spores with acacia gum on charcoal(CC1SA) formulation.
Apparent first-order kinetics of spore degradation at 608C (n),808C (l), 858C (!), and 908C (~), respectively. Points areexperimental data plotted according to Eq. 5, while lines arelinear regressions: R ( P) ¼ À0.61 (\0.1), À0.96 (\4 Â 10À4),À0.96 (\3 Â 10À4), À0.92 (\5 Â 10À4) for 60, 80, 85, and908C, respectively.
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temperature, the viability of spores in the control prepara-
tion, without carriers or acacia gum was only 6% (Table 2).
Release of spores
All preparations were tested for spore release from the
carriers. The graph in Figure 3 shows that spores are
released continuously, the number of free spores increasing
in time. The largest number of spores was released from the
formulations containing tapioca and CCþSA. Even after 7
weeks, the number of live spores in the supernatant of these
formulations was 2.6 to 4.0 Â 108 CFU/mL.
Discussion
We have found, as have others, that formulation and stor-
age conditions influence the viability of Bacillus spores.5
Compared with the other experimental conditions, unpro-
tected spores were the most sensitive to storage temperature.
Our data indicate that the rate of degradation, the loss of
spore viability, was highest without polymeric or carrier
Table 1. Thermodynamic Parameters Derived From the Arrhenius and Eyring Equations
Formulation Ea
(kcal/mol) A (1/day)D H
(kcal/mol)DS
[cal/(mol K)]T DS
(kcal/mol)DG, 608C(kcal/mol)
Tapioca þ spores 15.0 3.37 Â 108 14.5 0.8 2.7 14.2Tapioca þ spores in AG* 17.3 1.00 Â 1010 17.4 9.3 3.1 14.3Charcoal þ spores 18.7 1.60 Â 1010 18.0 8.4 2.8 15.2Charcoal with spores þ AG† 19.1 2.78 Â 1010 19.9 14.1 4.7 15.2Charcoal þ spores in AG‡ 17.3 2.96 Â 109 16.9 5.1 1.7 15.2Spores þ AG 11.4 1.33 Â 106 9.8 À13.8 À4.6 14.4Spores 7.1 6.58 Â 102 6.2 À25.9 À8.6 14.8
* Spores were first suspended in acacia gum (AG) and then combined with tapioca. † Charcoal was first loaded with spores and then coated with acaciagum. ‡ Spores were first suspended in acacia gum and then combined with charcoal.
Figure 2. Estimated viability of spores by Arrhenius and Eyring equations.
(a) Arrhenius plot. Points are experimental data plotted by Eq. 5, lines are linear regressions: R ( P) ¼ À0.96 (\0.04). (b) Eyring plot. Points are ex-perimental data plotted by Eq. 7 and lines are linear regressions. R ( P) ¼ À0.96 (\0.05). (c) Time for degradation of one log of spores at differenttemperatures. (d) Viability of spores at 58C; 1 - Tapioca þ spores, TS; 2 - Tapioca þ (spores þ acacia gum), TþSA; 3 - Charcoal þ spores, CCS;4 - (Charcoal þ spores) þ acacia gum, CCSþA; 5 - Charcoal þ (spores þ acacia gum), CCþSA; 6 - Spores þ acacia gum, SA; 7 - Spores, S.
Table 2. Predicted Viability of B. subtilis Spores in Charcoal andAcacia Gum (CCS1A Formulation) at Different Temperatures of
Storage in Comparison With Unprotected Spores (S)
Time
Viability of spores at different temperatures (%)
58C 208C 408C
CCSþA S CCSþA S CCSþA S
1 month 99.9 95.7 99.6 89.0 96.6 83.03 months 99.8 87.8 98.8 70.4 90.2 57.11 year 99.2 59.3 95.1 24.6 66.3 10.72 years 98.3 35.2 90.5 6.0 44.0 1.13 years 97.5 20.1 86.0 1.5 29.2 0.1
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protection. Acacia gum increased the viability of spores.
Combining the polymer of acacia gum with carriers such as
charcoal and tapioca augmented the viability of spores addi-
tionally. Consistent with these findings, the apparent activa-
tion energy, Ea, for degradation of unprotected spores was
smallest among all the spore preparations at 7.1 kcal/mol
(Table 1). This result is in close agreement with the findings
of Bruch and Smith,13 who estimated the Ea for Bacillus
spores on Teflon to be 9.5 kcal/mol. The unprotected spores
also showed the highest negative entropy change, DS, of
À25.9 cal/mol K (Table 1). When spores were dried in tapi-
oca and charcoal, the apparent Ea was increased and the T DS of degradation became positive. Even higher Ea was
achieved when porous carriers were combined with acacia
gum. The apparent Ea peaked at 19.9 kcal/mol at 333 K for
the spore formulations containing activated charcoal and sus-
pended in acacia gum.The energy associated with the change of entropy, T DS,
correlates with the change of enthalpy, D H , keeping the
Gibbs free energy, DG ¼ D H À T DS, minimal, often main-
taining it at a nearly constant. At T ¼ 333 K, in all prepara-
tions, the DG remains within 14–15 kcal/mol (Table 1 and
Figure 4a).
This thermodynamic phenomenon is known as enthalpy-
entropy compensation.14 The enthalpy-entropy compensation
phenomenon is characteristically displayed by a linear graph
of D H vs. DS as revealed in these studies when the experi-
mental results across different formulations were grouped as
in Figure 4b.
The change of enthalpy was positive,D
H [
0, for all for-mulations (Table 1) indicating that the process of spore deg-
radation is endothermic, i.e. it occurs with the absorption of
heat. The value of D H [ 0 for unprotected spores was rela-
tively small, equal to 6.2 kcal/mol. This value is comparable
to the D H [ 0 of 13 kcal/mol found for the unfolding of the
RNA from B. subtilis.15 In contrast, the D H [ 0 of the
Figure 4. Thermodynamic correlates of polymeric system.
(a) The energy associated with the change of entropy (T DS), the change of enthalpy (D H ), and the Gibbs free energy (DG ¼ D H À T DS) at T ¼333 K in samples of different formulations. (b) Enthalpy-entropy compensation for spore degradation in different formulations. Points are experimen-tal data, while a line is a linear regression: R ( P) ¼ 0.996 (\0.001). The line corresponds to the equation D H ¼ DGo þ T o DS, where DGo ¼ 1477kcal/mol and T o ¼ 342 K, the temperature is such that DS ¼ 0. (c) Relationship between parameters of the Arrhenius and Eyring equations (log Aand DS). Points are experimental data, while a line is a linear regression: R ( P) ¼ 0.990 (\0.0001). The line is described by Eq. 11. The experimen-tal slope of the line and Y -intersect, 0.19 Æ 0.2 cal/(mol K), (2 Æ 0.8) Â 108, respectively, compare well with 0.22 cal/(mol K) and 2.35 Â 108,respectively. (d) The apparent activation energy as function of the change of enthalpy as described by Eq. 10. Points are experimental data, and aline is a linear regression: R ( P) ¼ 0.993 (\0.0001). The experimental slope of the line, 0.9 Æ 0.5, compares well with the theoretical slope of the
line that is equal 1.0.
Figure 3. Release of spores from the different formulations intime.
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denaturation of proteins may have relatively higher values,
ranging from 20 to 5,300 kcal/mol.16
Low values for D H [0 does not particularize the involvement of a specific cellular
structural domain involved in the process of temperature-de-
pendent spore degradation. There are examples of important
cellular domains with low values for D H [ 0 of degradation
and/or denaturation.17,18 Immobilization of spores in acacia
gum alone significantly increased D H [ 0 from 6.2 to 9.8
kcal/mol. Associated with the T DS[ 0 of this formulation,the degradation of spores was significantly slower than that
of unprotected spores. Embedding spores in the porous car-
riers together with acacia gum was accompanied by even
higher values for D H [ 0, reaching its maximum at 19.9
kcal/mol for the activated charcoal with spores suspended in
acacia gum (Table 1). For the samples containing this for-
mulation, compared with unprotected spores at 58C, protec-
tion from degradation was increased by a factor of $70. As
predicted by Eq. 10, the values of the seven formulation
conditions for D Ea were followed closely by those for D H .Figure 4c demonstrates this good linear fit, R ¼ 0.99, P \0.0001.
T DS changed progressively from negative to increasingly
positive (Table 1) as protective conditions and the stability
of system were increased. When spores were immobilized in
acacia gum, T DS changed from À8.6 to 4.7 cal/(mol K). DS
\ 0 has conventionally been associated with decreases in
molecular rotational and translational degrees of freedom.
DS \ 0 in processes involving aqueous solvents often
involves the dehydration of solvent moieties forming the
interface between the contacting molecules and by release of
cations.19 The small positive or negative DS was observed
depended upon the competition between the DS[ 0 of sol-
vent dehydration and the DS \ 0 of the loss of molecular
translational and conformational degrees of freedom. We
speculate that acacia gum, though manifesting solvent dehy-
dration when dried and polymerized, traps some bound water thus preventing complete dehydration of the spore’s vital
components.
There was a good correspondence between observed val-
ues of the pre-exponential Arrhenius frequency factor, A,
and T DS that was predicted by Eq. 12 and confirmed in Fig-
ure 4d demonstrating a linear relationship between log A and
DS ( R ¼ 0.99, P \ 0.0001). Predicted and experimental
results portrayed in this graph are in a good agreement.
Our experiments showed that the release of spores, as for-
mulated in different carriers, increased in time and was in
the range of 107 – 108 CFU/mL even after 7 weeks of incu-
bation. These results are in good agreement with those
observed for the release of spores in 24 h from B. megate-rium pellets embedded in a variety of formulations.3
The
greatest release of B. subtilis spores was found in the for-
mulations with tapioca. The number of the spores released
after storage was increased when spores were treated with
acacia gum prior to immobilization in tapioca and charcoal.
Similar effect of acacia gum on the release of B. thuringien-
sis spores was observed for formulations containing various
polymers.7
Our investigations have shown that formulation of Bacil-
lus spores with acacia gum and tapioca and/or activated
charcoal as spore carriers significantly increased the shelf
life of the spores. Even after 2 years of storage at 208C the
formulations with acacia gum can protect 90% of spores,
while only 6% of unprotected spores remain alive at these
conditions. These findings are very important especially for
Bacillus probiotic strains because it shows the possibilities
for new probiotic formulations which can keep high rate of
strain viability even under ambient temperatures and do not
require cold chain during transportation.
New formulations with acacia gum together with charcoal
and tapioca cause as well the steady release of spores main-
taining their high viability over the 7-week experiments. It is
significant for improving formulations with Bacillus sporesrequired slow and consistent release of spores (for example,
in biopesticides, bioinsecticides). Thus, obtained results can
be used in the development of new formulations of Bacillus
spores for different biological products.
Conclusion
We found that new formulations with acacia gum and tap-
ioca or activated charcoal considerably increasing the shelf
life of Bacillus spores. For the spores embedded in gum aca-
cia, tapioca, or charcoal, we show that optimizing conditions
can extend spore life times by a factor of 60. Fundamental
Arrhenius and Eyring chemical rate equations, their tempera-
ture dependence, and Van’t Hoff relations that are usually
applied to compensation studies of organic chemical series
and aqueous macromolecular thermodynamics are used for
the analyses of a whole living cell system. We found that
increases in spore viability correlated with conjugate incre-
ments in enthalpy and entropy and negligible changes in
Gibbs free energy. The experimental results are consistent
with theoretically calculated data. These findings indicate the
possibilities for creating of new products, including Bacillusspores, with prolonged shelf-life and with slow and steady
release of spores. Theoretical and experimental approaches
presented in this study may be used for prediction of viabil-
ity and characterization of the degradation processes in bio-
logics for medicine and agriculture.
Acknowledgments
This research was supported by Aetos Technologies, Inc.,
and by FAA’s Office of Aerospace Medicine, as part of the Air
Transportation Center of Excellence for Airliner Cabin Envi-
ronment Research.
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Manuscript received Nov. 10, 2007, and revision received Apr. 17,2008.
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