1878 IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 38, NO. 8, AUGUST 2010 Cold Plasma Inactivation of Bacillus cereus and Bacillus anthracis (Anthrax) Spores Danil Dobrynin, Member, IEEE, Gregory Fridman, Member, IEEE, Yurii V. Mukhin, MeghanA. Wynosky-Dolfi, Judy Rieger, Richard F. Rest, Alexander F. Gutsol, and Alexander Fridman Abstract—Bacillus spores represent one of the most resistant organisms to conventional sterilization methods. This paper is focused on the inactivation of the spores of two Bacillus species, Bacillus cereus and Bacillus anthracis, using atmospheric- pressure dielectric-barrier-discharge (DBD) plasma. Spores treated in liquid or air-dried on a solid surface were effectively inactivated within 1 min of DBD plasma treatment at a discharge power of 0.3 W/cm 2 . Results of a series of model experiments show that neutral reactive oxygen species and UV radiation play a dominant role in the inactivation of spores. We also show that 45 s of the DBD plasma treatment of air-dried spores placed inside closed plastic or paper envelopes permits up to 7 log reduction of viable spores. Index Terms—Anthrax, atmospheric-pressure dielectric barrier discharge (DBD), Bacillus anthracis, nonequilibrium plasma, non- thermal plasma, spore inactivation, sterilization. I. I NTRODUCTION N ONTHERMAL atmospheric-pressure plasmas are inten- sively studied for possible use in various biological and medical applications. One of them is the inactivation of mi- croorganisms in water and air and on surfaces [1]–[7], including one of the most attractive applications of plasma—living-tissue sterilization [8]–[13]. Bacillus species, which are ubiquitous in the environment, are aerobic or facultative anaerobic gram- positive bacteria [14]–[16]. The genus Bacillus is divided into three broad groups, depending, among other characteristics, on the morphology of the spore. Bacillus cereus, Bacillus anthracis (anthrax), and Bacillus thuringiensis belong to the Bacillus cereus group [14], [17]. Moreover, morphological and Manuscript received August 26, 2009; revised November 15, 2009; accepted January 8, 2010. Date of publication March 22, 2010; date of current version August 11, 2010. This work was supported in part by the U.S. Department of Transportation under Grant PA-26-0017-01 and in part by the College of Medicine, Drexel University. D. Dobrynin is with the Electrical and Computer Engineering Department, College of Engineering, Drexel University, Philadelphia, PA 19104 USA (e-mail: danil.v.dobrynin@drexel.edu). G. Fridman is with the School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104 USA. Y. V. Mukhin and A. Fridman are with the Department of Mechanical Engineering and Mechanics, College of Engineering, Drexel University, Philadelphia, PA 19104 USA. M. A. Wynosky-Dolfi, J. Rieger, and R. F. Rest are with the Department of Microbiology and Immunology, College of Medicine, Drexel University, Philadelphia, PA 19129 USA. A. F. Gutsol was with the Department of Mechanical Engineering and Mechanics, College of Engineering, Drexel University, Philadelphia, PA 19104 USA. He is now with Chevron Energy Technology Company, Richmond, CA 94801-2016 USA. Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TPS.2010.2041938 chromosomal similarities between these species have prompted the view that Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus are all varieties of a single species [14]. Bacilli can produce a dormant cell type called a spore in response to nutrient-poor conditions. Bacterial spores have little or no metabolic activity and can withstand a wide range of environ- mental assaults including heat, UV, and solvents [18]–[21]. To kill or inactivate Bacillus spores, one can apply an 0.88-mol/L hydrogen peroxide at a pH of 5.0 for 3 h to sterilize a spore suspension of 10 6 spores/mL, or 10 6 rad of gamma irradiation to sterilize 10 6 spores/mL [21]. Bacillus anthracis spores, as opposed to vegetative cells, are the infectious form and cause anthrax. The spores of Bacillus anthracis represent a noteworthy bioterrorism agent and can be easily distributed in dry form in parcels and letters via postal service (as what occurred in 2001, when anthrax-contaminated letters sent through the U.S. postal service killed 5 people and sickened 23 others [22]), in aerosols, or in contaminated water, for instance. In response to these possibilities, an effective, low- energy, and cost-effective method of spore inactivation or ster- ilization is required. An attractive method of spore inactivation is plasma treatment. Low-temperature plasma at low pressure, arc discharge plasma, microwave plasma, and other plasmas are effective in the sterilization of spores [23]–[27]. For example, Kuo et al. reported that a 3–5 log reduction of Bacillus cereus spores in aqueous suspension can be achieved after several seconds of treatment with arc-seed microwave (2.45 GHz and 700 W) plasma torch [24]. Several systems based on different types of discharge have been reported [24]–[27]. In most of these systems, spores were treated either at low pressure or with relatively high power discharges, and 1–5 log reduction of germinated spores was achieved within a few minutes of treatment. In this paper, we were interested in inactivating Bacillus spores both in dry form and suspended in water with the use of atmospheric-pressure dielectric-barrier-discharge (DBD) plasma on surfaces as well as inside closed volumes, e.g., envelopes. We reported previously on the sterilization of bac- teria and yeast, including skin flora such as streptococcus and staphylococcus, on agar surfaces with atmospheric-pressure DBD plasma [28]. It took 5–10 s in the case of direct DBD treatment to achieve up to an 8 log reduction of a mixture of staphylococci, streptococci, and Candida yeast species. The results of this paper show that the inactivation of bacteria in spore form both in liquid or air-dried on surface requires higher doses of DBD plasma treatment, and up to 5 log reduction can be achieved within a minute of exposure to plasma. It is also 0093-3813/$26.00 © 2010 IEEE