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Biotechnol. Prog.

2008, 24, 1147À1153


Novel Methods for Storage Stability and Release of Bacillus Spores
Iryna B. Sorokulova, April A. Krumnow, and Suram Pathirana
Dept. of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849

Arnold J. Mandell
Dept. 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 Vodyanoy
Dept. 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 (

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 temperatures ranging from 408C to 908C and for bacterial release. Thermodynamic analysis showed that immobilization of spores in acacia gum significantly increased their viability compared 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 the spores 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 acacia gum and porous carriers (charcoal and tapioca) prolong the anticipated shelf-life of spores even 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

Bacteria of the Bacillus genus are usually considered to be very stable due to their formation of spores. The experimental data,1 however, indicate that only a few individual Bacillus spores from an initially large population can survive 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 producers of antibiotics, enzymes, aminoacids, and vitamins.2 Various Bacillus strains are known for their roles as probiotics 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
Correspondence concerning this article should be addressed to V. Vodyanoy at
C 2008 American Institute of Chemical Engineers V

numbers, Bacillus spores are frequently frozen, freezedried, 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 prolong the shelf life of spores.7 The two aims of this study are (1) to explore the effectiveness of preserving of Bacillus spores in new formulations using charcoal and tapioca as carriers along with acacia gum polymers to increase shelf-life and the slow and steady release of spores, and (2) to use thermodynamic characteristics 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.

The number of spores (colony forming units.8 Spore preparation A fresh. overnight culture of B. ¨ chner funnel with vacuum filtraSheybogan. Norway. subtilis spores. Decimal dilutions in sterile water were prepared and appropriative dilutions were plated onto NA (Difco. The spore formulations were put in 50-mL Falcon tubes (Becton Dickinson. Vol. dN ¼ ÀkN dt (1) where N is the concentration of spores being degraded. CþS: 75 mL of the spore suspension was diluted with 300 mL of sterile water and dried with 375 g of charcoal. A thermodynamic model of the thermal degradation of spores The apparent rate of degradation of spores during dehydration at constant temperature can be described by first-order kinetics Spore carriers and preparation of the protective biopolymer Activated charcoal (Sigma. Florence. k represents the first-order rate constant. subtilis ATCC 6051 was spread-plated on Difco Sporulation Medium. 4 yields log k ¼ À Ea 1 Â þ log A 2:303R T (5) If the logarithm of k in Eq. The moisture content in prepared formulations was determined by Karl Fisher titration. and 908C. time (t) yields an estimate of k from the slope. (Charcoal þ spores) þ acacia gum. they were heated at 808C for 20 min. 3 vs. WI) in a Bu tion and autoclaved at 1208C. Spores (S) and spores with acacia gum (SþA). MO) and granulated tapioca (Frontier. St. 5. Taking the logarithm of both sides of Eq.9 The plates were then incubated at 378C for 5 days. Rouen. subtilis spores in the formulations described above were tested for resistance to degradation at a range of temperatures. CFU) was counted for each time point and their mean and standard deviation were calculated in triplicate or more. Samples containing charcoal and tapioca were homogenized using the Tissue Tearor (model 985370. The solution was prepared by mixing the polymer powder with sterile water in concentrations of 15% (w/v). .837g for 40 min. the time of the thermal drying. Charcoal þ (spores þ acacia gum). Spores were then washed twice with sterile distilled water followed by centrifugation at 19. Racine. and t. Sparks. one obtains logðN =N0 Þ ¼ À2:303 Â kt (3) A plot of the left-hand side of Eq. 80. To eliminate vegetative cells. BD. 1/T . then the slope of this graph yields the activation energy (E a). Freshly prepared spores were used in all formulations. The solution of the equation (Eq. Charcoal þ spores. Louis. Spores were then stored in sterile distilled water at 48C until needed. 2. No. The solution was stirred and heated at 808C until the powder was completely dissolved. 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. Tapioca þ spores. 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. each containing 500 mg of the formulation. 75. MD). Sporulation was assessed by phase contrast microscopy which revealed $99% phase bright spores. 4. 65. and were used for the storage as a control.1148 Biotechnol. WI). Biospec Products. Vernon Hills. Franklin Lakes.10 N =N0 ¼ eÀkt (2) where N0 is the initial concentration of spores and N/N0 represents the ratio of the concentration of degraded spores to the initial spore concentration. Enumeration of viable spores was performed for all formulations before placing for storage and at the predetermined time intervals during the storage. prepared in the same ratio as in the formulations. Plates were then incubated at 378C for 18–24 h. 2008. 20 PSI for 15 min. 3. IL). Ea represents the apparent activation energy. the thermal activation level of transitions from spore viability to nonviability. cooled and stored at 48C. Tapioca þ (spores þ acacia gum). 5 subtilis is regarded as a model Gram-positive organism for genetic and physiological studies. 24. 5 is plotted against the reciprocal of temperature. The charcoal-spores preparation was then dried with the acacia gum solution. France) was used as the protective polymer. and A is the pre-exponential Arrhenius factor. NJ). Tubes with the varying formulations were placed in incubators at temperatures of: 40. 60. Heat resistance test for spore viability B. Five milliliters of cold sterile water were added to each tube and mixed thoroughly. 1) can be expressed as Formulation of spores in the carriers and polymer All the formulations were prepared using the Fluid Bed System (Applied Chemical Technology. Acacia gum (Colloids Naturels International. This fraction defines the ratio of viable of spores. Spores were collected by flooding the surface of the culture with sterile distilled water followed by scraping with a sterile cell spreader (Daigger.. Taking the logarithm of Eq. Prog. IA) were used as carriers for B. AL) at an outlet temperature of 708C: 1. CCSþA: 375 g of charcoal was mixed with 75 mL of the spore suspension. 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. TþS: 75 mL of the spore suspension was diluted with 300 mL of sterile water and dried with 375 g of tapioca. 55. 85. 2. The Arrhenius equation11 can be used to express the temperature dependence of the firstorder activation kinetics: k ¼ AeRT Ea (4) where R is the universal gas constant. It was then filtered using two Brew Rite1 coffee filters (Rockline Industries. 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.

21% Æ 0. DG is the standard Gibbs free energy of activation. 1/day for different formulations and the Arrhenius equation (Figure 2c). The Arrhenius and Eyring equations were then applied to the data in order to determine Ea. DH is the standard enthalpy of activation. 11. 5 1149 The rate constant of the spore degradation process depends on the thermodynamic activation parameters of this transition state and can be described by the Eyring equation 12 : k¼ kB T ÀDG KB T DS ÀDH e RT ¼ e R e RT h h (6) where k is the rate constant. 4 we can define the energy of activation as (8) each sample was removed. and DS of the temperature-dependent viability transitions. Taking the logarithm of both sides of Eq.. Points are experimental data plotted according to Eq. 2008.92 (\5 Â 10À4) for 60. Figure 1 shows the typical dependence 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. and 908C. The mean and standard deviation of the number of released spores were calculated for at least triplicate determinations. 1/T is linear. while lines are linear regressions: R (P) ¼ À0. 6. DG. 80.5%.1). These plots were then utilized for the calculation of Ea.01% in the products with tapioca to 1.96 (\3 Â 10À4). The highest viability was obtained for spores that were absorbed by charcoal and filled with acacia gum (Table 1. DS is the standard entropy of activation.5 aliquots of supernatant were removed at predetermined times. À0. The degradation rate followed first order kinetics in time across almost all temperatures. and 908C (~).06% in those containing activated charcoal.5 mL of sterile water was added to the tubes after . mixed and maintained at room temperature. 0. À0. varying from 0. The estimated time for spores to lose one log of their initial titers at different temperatures was calculated using the experimental values of the apparent rate constants of spore degradation in k. one obtains log A ¼ log   ekT DS þ h 2:303R (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. one obtains     k kB DS DH 1 À Â log þ ¼ log h T 2:303R 2:303R T (7) Figure 1. Before sampling for the determination of spore number. Stability of spores with both carriers was higher when acacia gum was added. Figure 2d).Biotechnol. 4) and the Eyring (Eq. h is Planck’s constant.9 Â 108 CFU/g. 6 and differentiation of this new expression with respect to 1/T shows that the Arrhenius (Eq. DH.2 Â 108 to 7. In this formulation. Prog. 858C (!). 5 and 7 and shown in the corresponding Figures 2a. The moisture content was low in all the formulations. kB is the Boltzmann constant. after 2 years of storage at 208C. ranging from 4. 3) was used to determine the best fits of the data with the apparent rate constant k. 9 with the k equivalent relation in Eq. respectively.96 (\4 Â 10À4). Vol. respectively. DG. 24. DH. At this The substitution of k in Eq.61 (\0. Enumeration of spore release Each sample consisting of 1 g of the indicated spore formulations was put in 50-mL Falcon tubes containing 10 mL of sterile water. and T is the absolute temperature in Kelvin. To maintain constant volume. DS from the ordinate intercept and the Gibbs free energy of activation by the relation: DG ¼ DH À T DS From Eq. The first-order kinetic model (Eq. 808C (l). A. Viability of spores with acacia gum on charcoal (CC1SA) formulation.b. À0. The plates were then incubated at 378C for 18–24 h. Those values are shown in Table 1. No. the tubes were mixed again and 0. If the plot of the left-hand side of Eq. 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.4% Æ 0. A. 85. @ ln k E a ¼ ÀR À 1 Á @ T (9) Results Characterization of the formulations The number of spores in the various formulations produced using the fluid bed system varied slightly. The apparent rate constants for spore degradation in the various formulations were used to obtain linear Arrhenius and Eyring plots constants with Eqs. one can compute the value of DH from the slope. the predicted viability of spores is 90. 7 vs. and DS. R is the gas constant. Apparent first-order kinetics of spore degradation at 608C (n). Serial 10-fold dilutions of each sample were prepared in sterile water and plated onto NA. 6) expressions can be related as Ea ¼ DH þ RT and (10)  A¼  ekB T DS eR h (11) Taking the logarithm of both sides of Eq. 5.

6 98. The graph in Figure 3 shows that spores are released continuously.5 408C CCSþA 96. (b) Eyring plot.0 Â 108 CFU/mL.Charcoal þ (spores þ acacia gum).1 A (1/day) 3. Table 1.2 14. Release of spores All preparations were tested for spore release from the carriers. 6 . the number of free spores increasing in time. that formulation and storage conditions influence the viability of Bacillus spores..Spores þ acacia gum.9 16.7 3.2 DS [cal/(mol K)] 0.78 2.3 44. SA.0 70.00 1.1 5. No.4 18. (c) Time for degradation of one log of spores at different temperatures. R (P) ¼ À0. 7 . Vol.7 1. the number of live spores in the supernatant of these formulations was 2.2 15.9 T DS (kcal/mol) 2. Our data indicate that the rate of degradation. CCSþA.2 98.1 17. Points are experimental data plotted by Eq.2 S 83.0 57.0 S 89.3 11.7 87.96 (\0. was highest without polymeric or carrier .Spores. 1 .5 S 95.8 9. subtilis Spores in Charcoal and Acacia Gum (CCS1A Formulation) at Different Temperatures of Storage in Comparison With Unprotected Spores (S) Viability of spores at different temperatures (%) 58C Time 1 3 1 2 3 month months year years years CCSþA 99.6 DG. CCS. as have others.6 À8. 608C (kcal/mol) 14.1 0. ‡ Spores were first suspended in acacia gum and then combined with charcoal. temperature. 2 . Table 2.7 19. Estimated viability of spores by Arrhenius and Eyring equations. 4 . lines are linear regressions: R (P) ¼ À0.0 17.8 59.8 6.7 À4. Predicted Viability of B.3 15.3 8.6 90. † Charcoal was first loaded with spores and then coated with acacia gum.96 (\0.2 14.8 À25.4 14.5 Compared with the other experimental conditions.2 15. the viability of spores in the control preparation. Points are experimental data plotted by Eq.8 99.37 1. TþSA.96 1.0 19.4 7.Charcoal þ spores.0 29.1150 Biotechnol. without carriers or acacia gum was only 6% (Table 2). 5.3 97.5 86.Tapioca þ spores. Prog.05). The largest number of spores was released from the formulations containing tapioca and CCþSA.2 20.8 95. Thermodynamic Parameters Derived From the Arrhenius and Eyring Equations Formulation Tapioca þ spores Tapioca þ spores in AG* Charcoal þ spores Charcoal with spores þ AG† Charcoal þ spores in AG‡ Spores þ AG Spores Ea (kcal/mol) 15. (a) Arrhenius plot.8 4. CCþSA.60 2.5 17.1 10.3 18. 7 and lines are linear regressions. (d) Viability of spores at 58C.58 Â Â Â Â Â Â Â 108 1010 1010 1010 109 106 102 DH (kcal/mol) 14. 2008. the loss of spore viability.2 66.7 1. 24.8 * Spores were first suspended in acacia gum (AG) and then combined with tapioca.33 6.1 90. S.9 9.9 99.0 1.1 À13. unprotected spores were the most sensitive to storage temperature.1 Discussion We have found. 5 Figure 2. TS. Even after 7 weeks.3 35.4 24.1 208C CCSþA 99.6 6.Tapioca þ (spores þ acacia gum).1 2.(Charcoal þ spores) þ acacia gum. 5 . 3 .04).4 14.6 to 4.

compares well with the theoretical slope of the line that is equal 1.9 cal/mol K (Table 1). for degradation of unprotected spores was smallest among all the spore preparations at 7. Thermodynamic correlates of polymeric system.. The apparent Ea peaked at 19. Points are experimental data. for all formulations (Table 1) indicating that the process of spore degradation is endothermic.14 The enthalpy-entropy compensation phenomenon is characteristically displayed by a linear graph of DH vs. Combining the polymer of acacia gum with carriers such as charcoal and tapioca augmented the viability of spores additionally. compare well with 0.5. 2008. 0.19 Æ 0. Consistent with these findings. When spores were dried in tapioca and charcoal. Even higher Ea was achieved when porous carriers were combined with acacia gum. (a) The energy associated with the change of entropy (T DS).15 In contrast. minimal.e.990 (\0.2 kcal/mol. 10. the apparent Ea was increased and the T DS of degradation became positive. the DG remains within 14–15 kcal/mol (Table 1 and Figure 4a). Points are experimental data. The value of DH [ 0 for unprotected spores was relatively small. The experimental slope of the line.Biotechnol. (2 Æ 0. This result is in close agreement with the findings of Bruch and Smith. 24. The experimental slope of the line and Y-intersect. T DS. No.9 Æ 0. in all preparations. DG ¼ DH À T DS. respectively. (c) Relationship between parameters of the Arrhenius and Eyring equations (log A and DS). often maintaining it at a nearly constant. The line corresponds to the equation DH ¼ DGo þ To DS. correlates with the change of enthalpy. i. Release of spores from the different formulations in time. 5 1151 protection. equal to 6.993 (\0.1 kcal/mol (Table 1). At T ¼ 333 K. . keeping the Gibbs free energy. the apparent activation energy. the temperature is such that DS ¼ 0. This thermodynamic phenomenon is known as enthalpyentropy compensation. DS as revealed in these studies when the experimental results across different formulations were grouped as in Figure 4b. (b) Enthalpy-entropy compensation for spore degradation in different formulations.22 cal/(mol K) and 2.8) Â 108. while a line is a linear regression: R (P) ¼ 0. The change of enthalpy was positive.001). The unprotected spores also showed the highest negative entropy change.996 (\0. This value is comparable to the DH [ 0 of 13 kcal/mol found for the unfolding of the RNA from B. Ea. The energy associated with the change of entropy.9 kcal/mol at 333 K for the spore formulations containing activated charcoal and suspended in acacia gum. Vol.0001). and the Gibbs free energy (DG ¼ DH À T DS) at T ¼ 333 K in samples of different formulations. (d) The apparent activation energy as function of the change of enthalpy as described by Eq.5 kcal/mol. Prog.2 cal/(mol K). subtilis.0. respectively. DH [ 0. spores on Teflon to be 9. DS. The line is described by Eq. 11. while a line is a linear regression: R (P) ¼ 0. Points are experimental data. DH. where DGo ¼ 1477 kcal/mol and To ¼ 342 K.35 Â 108. the change of enthalpy (DH). and a line is a linear regression: R (P) ¼ 0. Acacia gum increased the viability of spores. of À25. the DH [ 0 of the Figure 4.13 who estimated the Ea for Bacillus Figure 3. 0. it occurs with the absorption of heat.0001).

Markus A. reaching its maximum at 19. Fundamental Arrhenius and Eyring chemical rate equations. A. Kanjanamaneesathian M. with prolonged shelf-life and with slow and steady release of spores. Jantharangsri A. 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.16 Low values for DH [ 0 does not particularize the involvement of a specific cellular structural domain involved in the process of temperature-dependent spore degradation. Nilratana L. and by FAA’s Office of Aerospace Medicine. Literature Cited 1. the values of the seven formulation conditions for DEa were followed closely by those for DH. editors. while only 6% of unprotected spores remain alive at these conditions. Reich RR. J Parenteral Sci Technol.99. protection from degradation was increased by a factor of $70. thuringiensis spores was observed for formulations containing various polymers. Z Mikrobiol Epidemiol Immunobiol. Kivaev V. .7 Our investigations have shown that formulation of Bacillus spores with acacia gum and tapioca and/or activated charcoal as spore carriers significantly increased the shelf life of the spores. Theoretical and experimental approaches presented in this study may be used for prediction of viability and characterization of the degradation processes in biologics for medicine and agriculture. Origins Life Evol Biosphere. 1981. Dose K. and TDS that was predicted by Eq. The experimental results are consistent with theoretically calculated data. Figure 4c demonstrates this good linear fit. 12 and confirmed in Figure 4d demonstrating a linear relationship between log A and DS (R ¼ 0..300 kcal/mol. Bacillus stearothermophilus spore suspensions: effect of storage conditions and time on viability and moist heat resistance. 6. J Control Release.18 Immobilization of spores in acacia gum alone significantly increased DH [ 0 from 6. When spores were immobilized in acacia gum. 4. DNA stability and survival of Bacillus subtilis spores in extreme dryness. bioinsecticides). we show that optimizing conditions can extend spore life times by a factor of 60. or charcoal. 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. Predicted and experimental results portrayed in this graph are in a good agreement. Saltykov R. 2.0001. Even after 2 years of storage at 208C the formulations with acacia gum can protect 90% of spores.8 kcal/mol.17. megaterium pellets embedded in a variety of formulations. We found that increases in spore viability correlated with conjugate increments in enthalpy and entropy and negligible changes in Gibbs free energy. No.95:455–462. Wiwattanapatapee R. Thus. 1976.9 kcal/mol for the activated charcoal with spores suspended in acacia gum (Table 1). Ricca E. 1984. 3. 5. israelensis. 2004. 10.6:62–65. We speculate that acacia gum.7 cal/(mol K). viability and bacterial release studies.19:382–383. UK: Horizon Bioscience. including Bacillus spores. 5 denaturation of proteins may have relatively higher values. compared with unprotected spores at 58C.3 The greatest release of B. 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. These results are in good agreement with those observed for the release of spores in 24 h from B. London.1152 Biotechnol. Bakulov I. Floating pellets containing bacterial antagonist for control sheath blight of rice: formulations. increased in time and was in the range of 107 – 108 CFU/mL even after 7 weeks of incubation. Vol.2 to 9. R ¼ 0. Henriques O. Acknowledgments This research was supported by Aetos Technologies. the degradation of spores was significantly slower than that of unprotected spores. These findings indicate the possibilities for creating of new products. Gill M. Prog. tapioca. T DS changed progressively from negative to increasingly positive (Table 1) as protective conditions and the stability of system were increased. in biopesticides. 25:277–293. For the samples containing this formulation. 2008. as formulated in different carriers. P \ 0. For the spores embedded in gum acacia. P \ 0. Our experiments showed that the release of spores. There are examples of important cellular domains with low values for DH [ 0 of degradation and/or denaturation. Cutting SM. their temperature dependence. Effect of encapsulation on the persistence of Bacillus thuringiensis var. As predicted by Eq. DS \ 0 has conventionally been associated with decreases in molecular rotational and translational degrees of freedom.0001). Associated with the T DS [ 0 of this formulation..19 The small positive or negative DS was observed depended upon the competition between the DS [ 0 of solvent dehydration and the DS \ 0 of the loss of molecular translational and conformational degrees of freedom. Characteristics of the anthrax vaccinal strain STI-1 preserved for 30 years in the form of lyophilized spores. Embedding spores in the porous carriers together with acacia gum was accompanied by even higher values for DH [ 0. New formulations with acacia gum together with charcoal and tapioca cause as well the steady release of spores maintaining their high viability over the 7-week experiments. T DS changed from À8. Appl Microbiol Biotechnol. Conclusion We found that new formulations with acacia gum and tapioca or activated charcoal considerably increasing the shelf life of Bacillus spores. Bacterial Spore Formers: Probiotics and Emerging Applications. 24. Pelah Z. Matchavanich W. obtained results can be used in the development of new formulations of Bacillus spores for different biological products. There was a good correspondence between observed values of the pre-exponential Arrhenius frequency factor. Margalit J. 1995.99.35:74–78. Inc. ranging from 20 to 5. Gavrilov V. subtilis spores was found in the formulations with tapioca. It is significant for improving formulations with Bacillus spores required slow and consistent release of spores (for example. The number of the spores released after storage was increased when spores were treated with acacia gum prior to immobilization in tapioca and charcoal.6 to 4. Similar effect of acacia gum on the release of B. 2004. serotype H-14. Ulanova A. traps some bound water thus preventing complete dehydration of the spore’s vital components. Pengnoo A. as part of the Air Transportation Center of Excellence for Airliner Cabin Environment Research. though manifesting solvent dehydration when dried and polymerized.

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