Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus

Supercritical carbon dioxide (SC-CO₂) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO₂ treatment was tested using various conditions, such as pressure treatment (10–30 MPa), temperature (35–60 °C), and time (10–120 min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO₂ at 10 MPa and at 35 °C for 5 min. However, SC-CO₂ alone did not inactivate spores in biofilm even after the treatment time was extended to 120 min. When ethanol was used as a cosolvent with SC-CO₂ in the SC-CO₂ treatment using only 2–10 ml of ethanol in 100 ml of SC-CO₂ vessel for 60–90 min of treatment time at 10 MPa and 60 °C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO₂-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO₂ along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO₂ is efficient in inactivating the bacterial spores in the biofilm.