Utilization of low-pressure plasma to inactivate bacterial spores on stainless steel screws

Abstract

A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes.

FIG. 1.

Photograph (A) and scanning electron micrograph images (B–G) of a stainless steel screw contaminated with spores of B. pumilus strain SAFR-032 (B–F) and B. subtilis strain 168 (G). Scale bar: 2 mm (B), 1 mm (C), 100 μm (D), 25 μm (E), and 5 μm (F and G). Color images available online at www.liebertonline.com/ast

FIG. 2.

Sketch of the VHF-CCP used for sterilization experiments to measure inactivation kinetics of spores of B. pumilus strain SAFR-032 and B. subtilis strain 168 on stainless steel screws. On the left, a sketch of the grounded and the driven electrode is given, showing the electrical field. Color images available online at www.liebertonline.com/ast

FIG. 3.

Inactivation kinetics of spores of B. subtilis strain 168 (circles) and B. pumilus strain SAFR-032 (squares) on stainless steel screws obtained by direct plasma exposure [20 sccm hydrogen plasma, 400 W, 5 Pa (A)], vaporized 30% hydrogen peroxide treatment (B), and a two-step decontamination process of direct plasma exposure [20 sccm hydrogen plasma, 400 W, 5 Pa, and vaporized 30% hydrogen peroxide treatment (C)]. Data are expressed as averages and standard deviations (n=3). The open symbols indicate survival below the threshold of detection (i.e., complete spore reduction).