Inactivation of Vegetative and Sporulated Bacteria by Dry Gaseous Ozone

Abstract

Inactivation by gaseous ozone of different types of microorganisms is successfully achieved provided, as is well known, the gaseous phase is strongly humidified. The inactivation mechanisms and species involved in this process are, however, not yet clearly identified. To gain insight, we considered exposure of bacterial spores to dry rather than humidified ozone, a less complex chemical environment. In contrast to most of the published literature, it is shown that, under strict dry ozone conditions, bacterial spores can be inactivated, but to a degree that is largely dependent on the spore type and substrate material. In this case, the O₃ molecule is determined to be responsible for the inactivation process through its diffusion into and oxidative action within the spore, as no outer erosion of the spore is detected. With humidified ozone, a higher inactivation efficiency is observed that is most probably related, in part, to the swelling of the spore, which facilitates the diffusion of oxidative species within it and up to the core; besides O₃, these oxidative agents stem from the interaction of O₃ with H₂O, which in the end leads to a heavily damaged spore structure, in contrast to dry-ozone exposure where the spore integrity is maintained.

Keywords:

• Ozone
• Dry and Humidified Gaseous Ozone
• Microorganism Inactivation Mechanisms
• Vegetative Bacteria
• Sporulated Bacteria
• Spore Structural Damage

ACKNOWLEDGMENTS

The authors are grateful to Mr. J. S. Mayer for skilled technical assistance, A. Leduc for valuable assistance on enzymatic activity measurements and analysis, and Professor R. Hausler at École de Technologie Supérieure (ETS) for lending us the ozone generator. Thanks are also due to Professor B. Held from université de Pau and P. Renault from Air Liquide (France) for pertinent comments. Financial support was provided by the Fonds Québécois pour la Recherche sur la Nature et la Technologie (FQRNT), programme Équipe.

Notes

1. It corresponds to the Hartley molecular band (Ackerman, 1971; Pearce and Gaydon, 1976; Anderson et al., 1993; Brion et al., 1998; O'Keefe et al., 2005).

2. In contrast, dry gaseous ozone is known to be very effective to inactivate Gram positive and Gram negative bacteria (Da Silva et al., 1998), viruses (Emerson et al., 1982) and some other microorganisms.

3. Some authors consider that humidified ozone with RH values below 50% is “dry ozone”!

4. Within the group of “embedded” spores, only those with a morphologically well defined length were considered for the histogram, ignoring those where the extraneous material embedding the spore is clearly distinct from its body.

5. The Malachite green staining protocol involves heating the green dye to facilitate its eventual penetration into the spores. When the spore is not dormant, the green dye penetrates into the spores and is afterwards displaced by rinsing with water; an added red counter-stain can thus gain entry into the spore, which then becomes red in color.

6. Such an incubation delay is not observed with G. stearothermophilus when exposed, for instance, to the N₂-O₂ plasma afterglow in our lab.