Article pubs.acs.org/est
Inactivation of Aerosolized Bacillus atrophaeus (BG) Endospores and MS2 Viruses by Combustion of Reactive Materials Sergey A. Grinshpun,*,† Atin Adhikari,† Michael Yermakov,† Tiina Reponen,† Edward Dreizin,‡ Mirko Schoenitz,‡ Vern Hoffmann,‡ and Shasha Zhang‡ †
Center for Health-Related Aerosol Studies, Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267, United States ‡ Department of Chemical, Biological and Pharmaceutical Engineering, New Jersey Institute of Technology, Newark, New Jersey 07102, United States ABSTRACT: Accidental release of biological agents from a bioweapon facility may contaminate large areas, possibly causing disastrous environmental consequences. To address this issue, novel halogen-containing reactive materials are being designed with the added capability to inactivate viable airborne microorganisms. This study determined the efficiency of combustion products of such materials to inactivate aerosolized bacteria and viruses. Spores of Bacillus atrophaeus and MS2 viruses dispersed in dry air were exposed for subsecond time intervals to hydrocarbon flames seeded with different reactive powders so that bioaerosol particles interacted with the combustion products in a controlled high-temperature environment. The experiments were designed to quantify differences in the biocidal effects of different reactive material powders including Al and Mg, a B•Ti nanocomposite, an 8Al•MoO3 nanothermite, and a novel Al•I2 nanocomposite. Compared to pure hydrocarbon flame, powderseeded flame (with no iodine) produced about an order of magnitude greater inactivation of bacterial spores. The iodinecontaining material increased the spore inactivation by additional 2 orders of magnitude. The aerosolized MS2 viruses (generally not as stress-resistant as spores) were fully inactivated when exposed to combustion of either the iodinated or noniodinated powders. Overall, the study suggests a great biocidal potential of combustion products generated by novel iodine-containing nanocomposite materials.
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INTRODUCTION Inactivation of airborne microorganisms has a broad range of environmental applications, including (but not limited to) air purification, biohazard control, and aerobiology. The topic is particularly meaningful for the environmental protection associated with biodefense. Biological agents (such as bacterial spores or viruses) accidentally released from a bioweapon facility may be subjected to short- or long-range atmospheric transport. If some of the released microorganisms survive the environmental stress and remain pathogenic, they may contaminate large areas, potentially causing catastrophic damage to the public and infrastructure1 and posing significant environmental health challenges. The development of methods for effective destruction of aerosolized bioagents over a very short time period is among key priorities of the defense and environmental research programs in the U.S.2−4 and abroad.5,6 However, quantitative information on rapid (millisecond to second time range) inactivation of aerosolized bacterial spores and viruses is very scarce and mechanisms of such inactivation are not understood. Such mechanisms are expected to differ from those requiring much longer exposure times, e.g., germicidal irradiation or electrostatic fields. © 2012 American Chemical Society
This paper explores and quantifies rapid destruction of viable, stress-resistant, airborne microorganisms by exposing them to high-temperature combustion products of recently developed reactive materials, including halogen-containing reactive powders. Inactivation by halogens including iodine, chlorine, bromine, and fluorine or their compounds is considered to be one of the most effective methods.7 A material development approach was recently proposed, yielding novel filled nanocomposite materials (FNMs),8,9 comprising a metal matrix with inclusions of halogen or halogenated compound stabilized at low temperatures. The first FNM developed is an aluminum− iodine composite powder, which can be handled similarly to regular aluminum, but is capable of releasing iodine upon ignition. This material was prepared by mechanical milling of elemental aluminum and iodine at liquid nitrogen temperature. It was observed that adding iodine to aluminum accelerates ignition of metal, but somewhat extends the burn time of Received: Revised: Accepted: Published: 7334
February 10, 2012 May 30, 2012 June 4, 2012 June 4, 2012 dx.doi.org/10.1021/es300537f | Environ. Sci. Technol. 2012, 46, 7334−7341
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Figure 1. Experimental setup (general schematics).
individual particles.10 Because combustion is accompanied with iodine release, it is expected that the achieved combustion dynamics together with iodine-containing reaction products can substantially enhance inactivation of aerosolized stress-resistant bacterial spores and viruses exposed to combustion of the new material. Reliable detection of inactivation of aerosolized microorganisms is particularly challenging when the time of their exposure to biocidal materials is as short as a fraction of a secondthe condition relevant to a possible accidental release of biologically active aerosols. We have recently developed an experimental facility for studying how different environmental factors (heat, chemical reactive products, etc.) affect the viability of aerosolized microorganisms during a short-term ( HPQL in the most conservative estimate). A lower temperature threshold is needed for the complete inactivation of aerosolized MS2 viruses15 It is of a particular interest to define the near-wall portion of the flame-free zone in which Tgas ≤ 400 °C, i.e., some measurable survival is expected. We found that the width of this portion of the flame-free zone, δ, is 2.1 mm for the narrow flame (A), 0.8 mm for the intermediate case (B) and,
Figure 4. Gas temperature profiles in the flame free zone measured by a thermocouple inserted through the chamber wall for the narrow (A) and intermediate (B) flames. No powder is introduced to the flame.
exposure chamber and extends downstream creating a relatively narrow flame (A), a wider (intermediate) flame (B), and a flame wide enough to occupy the entire cross-section (C). In the case A, the flame is not only narrower but extends to a
Figure 5. Inactivation of aerosolized BG spores by combustion of different materials (narrow flame). 7338
dx.doi.org/10.1021/es300537f | Environ. Sci. Technol. 2012, 46, 7334−7341
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Figure 7. Inactivation of aerosolized MS2 viruses by combustion of different materials (narrow flame).
obviously, zero when the flame extends through the entire cross-section (C). Inactivation factors determined for airborne BG spores exposed to the three above-described configurations for the powder-free hydrocarbon flame were [GM (GSD)]: 50 (1.49) (A), 1205 (1.34) (B), and 26 852 (1.48) (C). As the flame-free cross-sectional area decreased, the IF value increased reaching about HPQL when the flame covered the entire cross-section, which confirms thatwithin the limit of quantification of the experimental protocolno spores survived inside the hydrocarbon flame. The exposure time was estimated to be ∼0.1 s (the characteristic longitudinal dimension divided to the average air velocity). At the same time, the spores may survive even at distances as short as a fraction of a millimeter from the edge of an open flame. The data suggest that a relatively small change in the flame dimensions inside the chamber configuration [e.g., (A) to (B) or (B) to (C)] may result in a sizable increase of IF: by 1−2 orders of magnitude in our tests. Using the temperature profiles shown in Figure 4 and the thermal inactivation experimental data reported in Grinshpun et al.,12 we estimated that the pure heat-induced inactivation (with no flame) in the chamber for cases (A) and (B) would be ∼90% (IF ∼10) and 99.9% (IF ∼1000), respectively. These estimates were, at least within an order of magnitude, in agreement with the experimentally determined values. The wide flame configuration produces somewhat trivial results. It does not allow differentiating between biocidal capabilities of different materials, which is the objective of this study. In contrast, the narrow flame configuration leaves an appreciable flame-free zone (Δ), including a sizable crosssectional area of relatively low “non-lethal” gas temperature (δ). Thus, it is advantageous from the practical standpoint to utilize the narrow flame design for comparative testing of different combustion formulations (e.g., iodinated versus noniodinated ones). The data on inactivation of BG spores challenged with combustion of different powders are presented in Figure 5. Combustion of noniodinated materials was found to produce inactivation factors [GM(GSD)] ranging from 231 (108) for 8Al•MoO3 to 857 (361) for Al3−4.5 μm . It was clearly seen that
the powder combustion (even without iodine and at moderate feeding rates) increases the spore “kill” greatly as compared to a pure hydrocarbon flame (which produced IF = 50 for this flame configuration). This difference was statistically significant (p < 0.01). No significant difference was identified between the spore inactivation factors generated by combustion of 8Al•MoO3 and Al10−14 μm (p > 0.05) Similarly, no significant difference in IF values was found for B•Ti, Mg, and Al3−4.5 μm (p > 0.05) The testing with an aluminum−iodine composite allowed to achieve a much more effective inactivation of airborne spores as compared to noniodinated powders: the observed enhancement was almost two decades (p < 0.01), and the inactivation factor exceeded 15 000 (shown in red in Figure 5). Figure 6 presents the optically measured flame temperatures of some of the tested reactive powders. These measurements showed the B•Ti nanocomposite as the powder burning hottest. At the same time, the temperature of the flame seeded with the Al•I2 nanocomposite was only marginally elevated above that of the flame seeded with unmodified aluminum of comparable size. These temperature observations do not correlate with the inactivation of the challenge aerosol shown in Figure 5. This shows that the enhanced inactivation of the iodine-bearing nanocomposite is not caused by a temperature effect but can rather be attributed to the release of iodinecontaining reaction products. The aerosolized MS2 viruses (which are generally not as stress-resistant as BG spores) were found to be effectively inactivated when exposed to combustion of both the iodinated and noniodinated powders as well as pure (nonseeded) hydrocarbon flame. The inactivation factors produced by all three formulations exceeded the quantification limit of our experimental protocol (see Figure 7). This finding indicates that MS2 viral particles did not serve well as a challenge bioaerosol for the present comparative testing of biocidal effects caused by combustion of different reactive materials. One major inactivation mechanism for BG spores can be mutational damage as demonstrated in our previous study.13 We have also discussed other dry heat induced inactivation mechanisms in our earlier publication.12 In brief, the DNA 7339
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damage, damage to essential proteins, and breaking of the coat layers by the inner pressure in bacterial endospore may all cause the spore viability loss. Damages to proteins and DNA are probably more important factors than breaking of the coat layers. For MS2 viruses, dry heat can directly damage (1) the coat protein, and/or (2) the A-protein, and/or (3) the coding regions on the single-stranded RNA. Each of these effects or all of them jointly can inactivate the MS2 virions and make it noninfectious to the host bacteria,31−34 which produces a lower PFU count. Iodine vapor can increase mutational frequency further or can make other changes in the bacterial or viral genetic material in the already mutated or structurally damaged spores or viruses. Hong and Ames,35 who conducted experiments with glycogen biosynthetic mutants of Salmonella typhimurium, stained asd+ transductants of this bacterium with iodine vapor under conditions favorable for bacteria making some glycogen. The investigators found that some transductants do not make glycogen (stained white) and some have other abnormalities (stained dark or blue). Clark and Pantoya36 reported that gaseous iodine can effectively interact with the spores and neutralizes bacterial growth. According to that study, oxides of iodine (e.g., I2O5) in combination with aluminum can be extremely effective at neutralizing bacterial endospores. However, because of a highly exothermic redox reaction occurring between Al and I2O5, such mixed materials may be difficult to handle, unlike the Al•I2 composites addressed in the present study. Overall, the study findings point to a great microbial inactivation potential of novel filled nanocomposite materials capable of releasing iodine during combustion. Furthermore, the study generated important information regarding the response of airborne viable bacterial spores and viruses to environmental stresses resulting from combustion of different materials.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: (513)558-0504; fax: (513)558-2263. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was funded through Grants HDTRA-1-08-1-0012 and HDTRA1-11-1-0017 from the Defense Threat Reduction Agency (DTRA, U.S. Department of Defense). The authors are grateful for this support.
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