Sterilization of Bacillus pumilus spores using supercritical fluid carbon dioxide containing various modifier solutions

Supercritical fluid carbon dioxide (SF-CO2) with small amounts of chemical modifier(s) provides a very effective sterilization technique that should be useful for destroying microorganism on heat-sensitive devices such as instruments flown on planetary-bound spacecraft. Under a moderate temperature (50 °C) and pressure (100 atm), spores of Bacillus pumilus strains ATCC 7061 and SAFR 032 can be effectively inactivated/eliminated from metal surfaces and small electronic devices in only 45 min using optimized modifier concentrations. Modifiers explored in this study included hydrogen peroxide (H2O2), tert-butyl hydroperoxide, formic acid, and Triton X-100. During sterilization procedure the modifiers were continuously added to SF-CO2 in either methanol or water at controlled concentrations. The lowest effective concentrations were established for each modifier. Complete elimination of both types of B. pumilus endospores occurred with an optimal modifier addition of either or 10% methanol containing 12% H2O2 or 12% tert-butyl hydroperoxide in SF-CO2, or a mixture of 6% H2O2 and 6% tert-butyl hydroperoxide. Using water as the carrier of SF-CO2 modifier, the complete elimination of spores viability of both B. pumilus strains occurred with an addition of either 3.3% water containing 3% H2O2, or 3.3% water containing 10% methanol and 0.5% formic acid, or 3.3% water containing 10% methanol, 1% formic acid and 2% H2O2.